US20090053786A1 - Prevention of disulfide bond reduction during recombinant production of polypeptides - Google Patents

Prevention of disulfide bond reduction during recombinant production of polypeptides Download PDF

Info

Publication number
US20090053786A1
US20090053786A1 US12/217,745 US21774508A US2009053786A1 US 20090053786 A1 US20090053786 A1 US 20090053786A1 US 21774508 A US21774508 A US 21774508A US 2009053786 A1 US2009053786 A1 US 2009053786A1
Authority
US
United States
Prior art keywords
antibodies
antibody
thioredoxin
human
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/217,745
Other languages
English (en)
Inventor
Yung-Hsiang Kao
Michael W. Laird
Melody Trexler Schmidt
Rita L. Wong
Daniel P. Hewitt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genentech Inc
Original Assignee
Genentech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=39791340&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20090053786(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to US12/217,745 priority Critical patent/US20090053786A1/en
Application filed by Genentech Inc filed Critical Genentech Inc
Assigned to GENENTECH, INC. reassignment GENENTECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHMIDT, MELODY TREXLER, KAO, YUNG-HSIANG, HEWITT, DANIEL P., LAIRD, MICHAEL W., WONG, RITA L.
Publication of US20090053786A1 publication Critical patent/US20090053786A1/en
Priority to US13/354,223 priority patent/US8574869B2/en
Priority to US14/043,758 priority patent/US20140128575A1/en
Priority to US15/488,917 priority patent/US20170313780A1/en
Priority to US16/240,592 priority patent/US10759866B2/en
Priority to US16/847,317 priority patent/US10906986B2/en
Priority to US16/847,309 priority patent/US10808037B1/en
Priority to US17/087,313 priority patent/US11639395B2/en
Priority to US17/124,314 priority patent/US11078294B2/en
Priority to US18/194,471 priority patent/US20230383004A1/en
Priority to US18/310,448 priority patent/US20230365704A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the invention concerns methods and means for preventing the reduction of disulfide bonds during the recombinant production of disulfide-containing polypeptides.
  • the invention concerns the prevention of disulfide bond reduction during harvesting of disulfide-containing polypeptides, including antibodies, from recombinant host cell cultures.
  • recombinant proteins are produced by cell culture, using either eukaryotic cells, such as mammalian cells, or prokaryotic cells, such as bacterial cells, engineered to produce the protein of interest by insertion of a recombinant plasmid containing the nucleic acid encoding the desired protein.
  • eukaryotic cells such as mammalian cells
  • prokaryotic cells such as bacterial cells
  • 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.
  • Mammalian cells have become the dominant system for the production of mammalian proteins for clinical applications, primarily due to their ability to produce properly folded and assembled heterologous proteins, and their capacity for post-translational modifications.
  • Chinese hamster ovary (CHO) cells, and cell lines obtained from various other mammalian sources such as, for example, mouse myeloma (NS0), baby hamster kidney (BHK), human embryonic kidney (HEK-293) and human retinal cells, such as the PER.C6® cell line isolated from a human retinal cell, which provides human glycosylation characteristics, and is able to naturally produce antibodies that match human physiology, have been approved by regulatory agencies for the production of biopharmaceutical products.
  • a small number of transformed recombinant host cells are allowed to grow in culture for several days (see, e.g., FIG. 23 ). Once the cells have undergone several rounds of replication, they are transferred to a larger container where they are prepared to undergo fermentation.
  • the media in which the cells are grown and the levels of oxygen, nitrogen and carbon dioxide that exist during the production cycle may have a significant impact on the production process. Growth parameters are determined specifically for each cell line and these parameters are measured frequently to assure optimal growth and production conditions.
  • the recombinant protein can be harvested.
  • the cells are engineered to secrete the polypeptide into the cell culture media, so the first step in the purification process is to separate the cells from the media.
  • harvesting includes centrifugation and filtration to produce a Harvested Cell Culture Fluid (HCCF).
  • HCCF Harvested Cell Culture Fluid
  • the media is then subjected to several additional purification steps that remove any cellular debris, unwanted proteins, salts, minerals or other undesirable elements.
  • the recombinant protein is highly pure and is suitable for human therapeutic use.
  • the instant invention generally relates to a method for preventing reduction of a disulfide bond in a polypeptide expressed in a recombinant host cell, comprising supplementing the pre-harvest or harvested culture fluid of the recombinant host cell with an inhibitor of thioredoxin or a thioredoxin-like protein.
  • the thioredoxin inhibitor is added to the pre-harvest culture fluid.
  • the thioredoxin inhibitor is added to the harvested culture fluid.
  • the thioredoxin inhibitor is a direct inhibitor of thioredoxin.
  • the thioredoxin inhibitor may, for example, be an alkyl-2-imidazolyl disulfide or a naphthoquinone spiroketal derivative.
  • the thioredoxin inhibitor is a specific inhibitor of thioredoxin reductase.
  • the thioredoxin inhibitor is a gold complex, where the gold complex may, for example, be aurothioglucose (ATG) or aurothiomalate (ATM). While the effective inhibitory concentration may vary, it typically is between about 0.1 mM and 1 mM. Similarly, the minimum effective inhibitory concentration varies depending on the nature of the polypeptide and overall circumstances, and is typically reached when the ATG or ATG concentration is at least about four-times of thioreduxin concentration in the pre-harvest or harvested culture fluid.
  • ATG aurothioglucose
  • ATM aurothiomalate
  • the thioredoxin inhibitor is a metal ion, where the metal ion, without limitation, may be selected from the group consisting of Hg 2+ , Cu 2+ , Zn 2+ , Co 2+ , and Mn 2+ .
  • the effective inhibitory concentration generally is between about 5 ⁇ M and about 100 ⁇ M, or between about 10 ⁇ M and about 80 ⁇ M, or between about 15 ⁇ M and about 50 ⁇ M.
  • the minimum inhibitory concentration of cupric sulfate also varies, but typically is reached when cupric sulfate is added at a concentration at least about two-times of thioredoxin concentration in the pre-harves or harvested culture fluid.
  • the thioredoxin inhibitor is an oxidizing agent, e.g., an inhibitor of G6PD, such as, for example, pyridoxal 5′-phosphate, 1 fluoro-2,4 dinitrobenzene, dehydroepiandrosterone (DHEA) or epiandrosterone (EA); cystine or cysteine.
  • an inhibitor of G6PD such as, for example, pyridoxal 5′-phosphate, 1 fluoro-2,4 dinitrobenzene, dehydroepiandrosterone (DHEA) or epiandrosterone (EA); cystine or cysteine.
  • DHEA dehydroepiandrosterone
  • EA epiandrosterone
  • cystine or cysteine cysteine.
  • Typical effective inhibitor concentrations of DHEA are between about 0.05 mM and about 5 mM, or between about 0.1 mM and about 2.5 mM.
  • the thioredoxin inhibitor is an inhibitor of hexokinase activity, including, without limitation, chelators of metal ions, such as, for example, ethylenediamine tetraacetic acid (EDTA).
  • EDTA is typically added and effective at a concentration between about 5 mM and about 60 mM, or about 10 mM and about 50 mM, or about 20 mM and about 40 mM.
  • the inhibitor of hexokinase activity is selected from the group consisting of sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose, and lyxose.
  • inhibitors include cystine, cysteine, and oxidized glutathione which are typically added at a concentration at least about 40-times of the concentration of the polypeptide in question in the pre-harvest or harvested culture fluid.
  • the thioredoxin inhibitor is an siRNA, an antisense nucleotide, or an antibody specifically binding to a thioredoxin reductase.
  • the thioredoxin inhibitor is a measure indirectly resulting in the inhibition of thioredoxin activity.
  • This embodiment includes, for example, air sparging the harvested culture fluid of the recombinant host cell, and/or lowering the pH of the harvested culture fluid of the recombinant host cell.
  • indirect means for inhibiting thioredoxin activity such as air sparging and/or lowering of the pH, can be combined with the use of direct thioredoxin inhibitors, such as those listed above.
  • the polypeptide may, for example, be an antibody, or a biologically functional fragment of an antibody.
  • Representative antibody fragments include Fab, Fab′, F(ab′) 2 , scFv, (scFv) 2 , dAb, complementarity determining region (CDR) fragments, linear antibodies, single-chain antibody molecules, minibodies, diabodies, and multispecific antibodies formed from antibody fragments.
  • Therapeutic antibodies include, without limitation, 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 ⁇ 4 ⁇ 7 integrin antibodies; anti-EGFR antibodies; anti-CD3 antibodies; anti-CD25 antibodies; anti-CD4 antibodies; anti-CD52 antibodies; anti-Fc receptor antibodies; anti-carcinoembryonic antigen (CEA) antibodies; antibodies directed against breast epithelial cells; antibodies that bind to colon carcinoma cells; anti-CD38 antibodies; anti-CD33 antibodies; anti-CD22 antibodies; anti-EpCAM antibodies; anti-GpIIb/IIIa antibodies; anti-RSV antibodies; anti-CMV antibodies; anti-HIV antibodies; anti-hepatitis antibodies; anti-CA 125 antibodies; anti- ⁇ v ⁇ 3 antibodies; anti-human renal cell carcinoma antibodies; anti-human 17-1A
  • the therapeutic antibody is an antibody binding to a HER receptor, VEGF, IgE, CD20, CD11a, CD40, or DR5.
  • the HER receptor is HER1 and/or HER2, preferably HER2.
  • the HER2 antibody may, for example, comprise a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, and 19.
  • the therapeutic antibody is an antibody that binds to CD20.
  • the anti-CD20 antibody may, for example, comprise a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 1 through 15.
  • the therapeutic antibody is an antibody that binds to VEGF.
  • the anti-VEGF antibody may, for example, comprise a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 20 through 25.
  • the therapeutic antibody is an antibody that binds CD11a.
  • the anti-CD11a antibody may, for example, comprise a heavy and/or light chain variable domain sequence selected from the group consisting of SEQ ID NOS: 26 through 29.
  • the therapeutic antibody binds to a DR5 receptor.
  • the anti-DR5 antibody may, for example, be selected from the group consisting of Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and 25.3, and preferably is Apomab 8.3 or Apomab 7.3, and most preferably Apomab 7.3.
  • the polypeptide expressed in the recombinant host cell is a therapeutic polypeptide.
  • the therapeutic polypeptide can be selected from the group consisting of a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; en
  • the recombinant host cell can be an eukaryotic host cell, such as a mammalian host cell, including, for example, Chinese Hamster Ovary (CHO) cells.
  • a mammalian host cell including, for example, Chinese Hamster Ovary (CHO) cells.
  • the recombinant host cell can also be a prokaryotic host cell, such as a bacterial cell, including, without limitation, E. coli cells.
  • FIG. 1 Dialysis Experiment: Digital gel-like imaging obtained from Bioanalyzer analysis (each lane representing a time point) demonstrating that ocrelizumab (rhuMAb 2H7—Variant A) inside the dialysis bag remained intact during the incubation period.
  • FIG. 2 Dialysis Experiment: Digital gel-like imaging obtained from Bioanalyzer analysis (each lane representing a time point) showing that ocrelizumab outside the dialysis bag was reduced during the incubation period. This is evidenced by the loss of intact antibody ( ⁇ 150 kDa) and the formation of antibody fragments depicted in the Figure. At the 48-hour time point (Lane 7), the reduced antibody appeared to be reoxidized, presumably as a result of loosing reduction activity in the Harvested Cell Culture Fluid (HCCF). The band appearing just above the 28 kDa marker arose from the light chain of antibody. There was a significant amount of free light already present in the HCCF before the incubation began. The presence of excess free light chain and dimers of light chain in the HCCF is typical for the cell line producing ocrelizumab.
  • HCCF Harvested Cell Culture Fluid
  • FIG. 3 Free Thiol Levels from Dialysis Experiment: Purified ocrelizumab in phosphate buffered saline (PBS) was inside the dialysis bag and HCCF containing ocrelizumab was outside the bag. Free thiols inside (boxes) and outside (diamonds) the dialysis bag reached comparable levels within a few hours, indicating a good exchange of small molecule components in the HCCF between inside and outside the dialysis bag.
  • PBS phosphate buffered saline
  • FIG. 4 Thioredoxin System and Other Reactions Involved in Antibody Reduction:
  • the thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, functions as a hydrogen donor system for reduction of disulfide bonds in proteins.
  • Trx is a small monomeric protein with a CXXC active site motif that catalyzes many redox reactions through thiol-disulfide exchange.
  • the oxidized Trx can be reduced by NADPH via TrxR.
  • the reduced Trx is then able to catalyze the reduction of disulfides in proteins.
  • the NADPH required for thioredoxin system is provided via reactions in pentose phosphate pathway and glycolysis.
  • FIG. 5 In Vitro Activity of Thioredoxin System: Digital gel-like image from Bioanalyzer analysis (each lane representing 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 the complete reduction of ocrelizumab; the ocrelizumab was completely reduced in less than 21 hours.
  • FIG. 6 In Vitro Activity of Thioredoxin System Inhibited by Aurothioglucose: The addition of aurothioglucose (ATG) to the same reaction mixture as described in the caption for FIG. 5 , above, effectively inhibited the ocrelizumab reduction. This is seen by the digital gel-like image from Bioanalyzer analysis (each lane representing a time point).
  • Aurothioglucose aurothioglucose
  • FIG. 7 In vitro Activity of Thioredoxin System Inhibited by Aurothiomalate: The addition of aurothiomalate (ATM) at a concentration of 1 mM to the same reaction mixture as described in the caption for FIG. 5 , above, effectively inhibited the ocrelizumab reduction. This is seen by the digital gel-like image from Bioanalyzer analysis (each lane representing a time point).
  • ATM aurothiomalate
  • FIG. 8 In Vitro Activity of Thioredoxin System: Digital gel-like image from Bioanalyzer analysis (each lane representing 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.
  • FIG. 9 In vitro Activity of Thioredoxin System Inhibited by CuSO 4 : The addition of CUSO 4 at a concentration of 50 ⁇ M to the same reaction mixture as described in the caption for FIG. 8 effectively inhibited the ocrelizumab reduction as shown in the digital gel-like image from Bioanalyzer analysis (each lane representing a time point).
  • FIG. 10 Ocrelizumab Reduction: Digital gel-like image from Bioanalyzer analysis (each lane representing a time point) showing that ocrelizumab was reduced in an incubation experiment using HCCF from a homogenized CCF generated from a 3-L fermentor.
  • FIG. 11 Inhibition of Ocrelizumab Reduction In HCCF by Aurothioglucose: Digital gel-like image from Bioanalyzer analysis (each lane representing a time point) showing that the addition of 1 mM aurothioglucose to the same HCCF as used for the incubation experiment as shown in FIG. 10 inhibited the reduction of ocrelizumab.
  • FIG. 12 Inhibition of Ocrelizumab Reduction In HCCF by Aurothiomalate: Digital gel-like image from Bioanalyzer (each lane representing a time point) analysis indicating that the addition of 1 mM aurothiomalate to the same HCCF as used for the incubation experiment shown in FIG. 10 inhibited the reduction of ocrelizumab.
  • FIG. 13 Losing Reduction Activity in HCCF: The HCCF from one of the large scale manufacturing runs for ocrelizumab (the “beta” run) that was subject to several freeze/thaw cycles demonstrated no ocrelizumab reduction when used in an incubation experiment. This was shown by Bioanalyzer analysis (each lane representing a time point), and can be contrasted to the antibody reduction seen previously in the freshly thawed HCCF from the same fermentation batch.
  • FIG. 14 The Lost Reduction Activity in HCCF Restored by Addition of NADPH: The reduction of ocrelizumab was observed again in the Bioanalyzer assay (each lane representing a time point) after the addition of NADPH at a concentration of 5 mM into the HCCF where the reduction activity has been eliminated under the conditions described above in FIG. 13 .
  • FIG. 15 The Lost Reduction Activity in HCCF Restored by Addition of Glucose-6-Phosphate: The reduction of ocrelizumab was observed again in the Bioanalyzer assay (each lane representing a time point) after the addition of G6P at a concentration of 10 mM into the HCCF where the reduction activity has been eliminated due to the treatment described above in FIG. 13 .
  • FIG. 16 Ocrelizumab Reduction: A digital gel-like image from Bioanalyzer analysis showing that ocrelizumab was reduced in an incubation experiment using a HCCF from a large scale manufacturing run (the “alpha” run).
  • FIG. 17 EDTA Inhibits Ocrelizumab Reduction: Digital gel-like image from Bioanalyzer analysis (each lane representing a time point) showing that the reduction of ocrelizumab was inhibited in an incubation experiment using a HCCF from the alpha run with EDTA added at a concentration of 20 mM to the HCCF whose reducing activity is demonstrated in FIG. 16 .
  • FIG. 18 The Lost Reduction Activity in “Beta Run” HCCF Restored by Addition of Glucose-6-Phosphate but No Inhibition of Reduction by EDTA: The reduction of ocrelizumab was observed in the Bioanalyzer assay (each lane representing a time point) after the addition of G6P at a concentration of 5 mM and 20 mM EDTA into the HCCF whose reduction activity had been lost (see FIG. 13 ). In contrast to the results shown in FIG. 17 , the presence of EDTA did not block the reduction of ocreliumab.
  • FIG. 19 Inhibition of Ocrelizumab Reduction: by (i) addition of EDTA, (ii) addition of CuSO 4 , or (iii) adjustment of pH to 5.5. All three different methods, (1) addition of EDTA, (2) addition of CuSO 4 , and (3) adjustment of pH to 5.5, used independently, were effective in inhibiting ocrelizumab reduction. This was demonstrated by the depicted quantitative Bioanalyzer results that showed that nearly 100% intact (150 kDa) antibody remained in the protein A elution pools. In contrast, ocrelizumab was completely reduced in the control HCCF after 20 hours of HCCF hold time.
  • FIG. 20 Inhibition of Ocrelizumab Reduction by Air Sparging: Sparging the HCCF with air was effective in inhibiting ocrelizumab disulfide bond reduction. This was demonstrated by the quantitative Bioanalyzer results showing that nearly 100% intact (150 kDa) antibody remained in the protein A elution pools. In contrast, ocrelizumab was almost completely reduced in the control HCCF after 5 hours of sparging with nitrogen.
  • FIG. 21 shows the V L (SEQ ID NO. 24) amino acid sequence of an anti-Her2 antibody (Trastuzumab).
  • FIG. 22 shows the V H (SEQ ID No. 25) amino acid sequence of an anti-Her2 antibody (Trastuzumab).
  • FIG. 23 is a schematic showing some steps of a typical large scale manufacturing process.
  • FIG. 24 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate.
  • FIG. 25 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 1 mM histidine sulfate+1 mM ATG.
  • FIG. 26 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+0.6 ⁇ M ATG (6:1 ATG:TrxR).
  • FIG. 27 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+0.4 ⁇ M ATG (4:1 ATG:TrxR).
  • FIG. 28 is a digital gel-like image from Bioanalyzer analysis: 2H7. (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+0.2 ⁇ M ATG (2:1 ATG:TrxR).
  • FIG. 29 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+0.1 mM autothiomalate (ATM).
  • FIG. 30 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+0.01 mM autothiomalate (ATM).
  • FIG. 31 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+20 ⁇ M CUSO 4 (4:1 Cu 2+ :Trx).
  • FIG. 32 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+10 ⁇ M CUSO 4 (2:1 Cu 2+ :Trx).
  • FIG. 33 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+5 ⁇ M CuSO 4 (1:1 Cu 2+ :Trx).
  • FIG. 34 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+532 ⁇ M cystamine (20:1 cystamine:2H7 disulfide).
  • FIG. 35 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+266 ⁇ M cystamine (10:1 cystamine:2H7 disulfide).
  • FIG. 36 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+133 ⁇ M cystamine (5:1 cystamine:2H7 disulfide).
  • FIG. 37 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+26.6 ⁇ M cystamine (1:1 cystamine:2H7 disulfide).
  • FIG. 39 is a digital gel-like image from Bioanalyzer analysis: 2H7 (Variant A)+1 mM NADPH+5 ⁇ M thioredoxin+0.1 ⁇ M thioredoxin reductase (recombinant) in 10 mM histidine sulfate+2.6 mM GSSG (oxidized glutathione).
  • the term “reduction” is used to refer to the reduction of one or more disulfide bonds of the protein or antibody.
  • the terms “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 reuction.”
  • reduction or “disulfide bond reduction” are used in the broadest sense, and include complete and partial reduction and reduction of some or all of the disulfide bonds, interchain or intrachain, present in a protein such as an antibody.
  • protein is meant a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” or other small molecular weight drugs that do not have such structure.
  • the protein herein will have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD.
  • proteins encompassed within the definition herein include all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds.
  • therapeutic protein or “therapeutic polypeptide” refers to a protein that is used in the treatment of disease, regardless of its indication or mechanism of action. In order for therapeutic proteins to be useful in the clinic it must be manufactured in large quantities.
  • Manufacturing scale production of therapeutic proteins, or other proteins, utilize cell cultures ranging from about 400 L to about 80,000 L, depending on the protein being produced and the need. Typically such manufacturing scale production utilizes cell culture sizes from about 400 L to about 25,000 L. Within this range, specific cell culture sizes such as 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000 L, about 14,000 L, or about 16,000 L are utilized.
  • therapeutic antibody refers to an antibody that is used in the treatment of disease.
  • a therapeutic antibody may have various mechanisms of action.
  • a therapeutic antibody may bind and neutralize the normal function of a target associated with an antigen.
  • a monoclonal antibody that blocks the activity of the of protein needed for the survival of a cancer cell causes the cell's death.
  • Another therapeutic monoclonal antibody may bind and activate the normal function of a target associated with an antigen.
  • a monoclonal antibody can bind to a protein on a cell and trigger an apoptosis signal.
  • Yet another monoclonal antibody may bind to a target antigen expressed only on diseased tissue; conjugation of a toxic payload (effective agent), such as a chemotherapeutic or radioactive agent, to the monoclonal antibody can create an agent for specific delivery of the toxic payload to the diseased tissue, reducing harm to healthy tissue.
  • a toxic payload such as a chemotherapeutic or radioactive agent
  • a “biologically functional fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least specific binding to the target antigen.
  • diagnosis protein refers to a protein that is used in the diagnosis of a disease.
  • diagnostic antibody refers to an antibody that is used as a diagnostic reagent for a disease.
  • the diagnostic antibody may bind to a target antigen that is specifically associated with, or shows increased expression in, a particular disease.
  • the diagnostic antibody may be used, for example, to detect a target in a biological sample from a patient, or in diagnostic imaging of disease sites, such as tumors, in a patient.
  • a “biologically functional fragment” of a diagnostic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least specific binding to the target antigen.
  • “Purified” means that a molecule is present in a sample at a concentration of at least 80-90% by weight of the sample in which it is contained.
  • the protein, including antibodies, which is purified is preferably essentially pure and desirably essentially homogeneous (i.e. free from contaminating proteins etc.).
  • An “essentially pure” protein means a protein composition comprising at least about 90% by weight of the protein, based on total weight of the composition, preferably at least about 95% by weight.
  • An “essentially homogeneous” protein means a protein composition comprising at least about 99% by weight of protein, based on total weight of the composition.
  • the protein is an antibody.
  • Antibodies (Abs) and “immunoglobulins” (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single-chain molecules such as scFv molecules, as well as antibody fragments (e.g., Fab, F(ab′) 2 , and Fv).
  • a monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • 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.
  • the monoclonal antibodies to be used in accordance with the present invention may be made 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.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the humanized antibody includes a PrimatizedTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • affinity matured antibody is one with one or more alterations in one or more CDRs/HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • Affinity matured antibodies are produced by procedures known in the art. Marks et al., Bio/Technology 10:779-783 (1992) describes affinity maturation by V H and V L domain shuffling. Random mutagenesis of CDR/HVR and/or framework residues is described by: Barbas et al., Proc Nat. Acad. Sci.
  • variable region refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • variable domain of the heavy chain may be referred to as “V H .”
  • variable domain of the light chain may be referred to as “V L .” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR).
  • CDRs complementarity-determining regions
  • HVRs hypervariable regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest , Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequences of their constant domains.
  • antibodies can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 .
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.
  • An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
  • full length antibody “intact antibody” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below.
  • Antibody fragments comprise only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody.
  • an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • an antibody fragment for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding.
  • an antibody fragment is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
  • such an antibody fragment may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • 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 yields an F(ab′) 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • the Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab′ fragments differ from Fab fragments by the addition of a few residues at the carboxy 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 residue(s) of the constant domains bear a free thiol group.
  • F(ab′)2 antibody fragments originally were produced as pairs of Fab′ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • Fv is the minimum antibody fragment which contains a complete antigen-binding site.
  • a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association.
  • scFv single-chain Fv
  • one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the V H —V L dimer.
  • the six CDRs confer antigen-binding specificity to the antibody.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H —V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; 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.
  • the antibody may bind to any protein, including, without limitation, a member 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 av/p3 integrin including either ⁇ or ⁇ or subunits thereof (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.
  • HER1 EGFR
  • HER2, HER3 and HER4 CD proteins
  • cell adhesion molecules such as LFA-1, Mol, p150
  • 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; lipoproteins; ⁇ -1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or tissue-type plasminogen activator (t-PA); bombazine; thrombin; tumor necrosis factor- ⁇ and - ⁇ ; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1- ⁇ ); serum albumin such as human serum albumin (HSA); mullerian
  • a “biologically functional fragment” of an antibody comprises only a portion of an intact antibody, wherein the portion retains at least one, and as many as most or all, of the functions normally associated with that portion when present in an intact antibody.
  • a biologically functional fragment of an antibody comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen.
  • a biologically functional fragment of an antibody for example one that comprises the Fc region, retains at least one of the biological functions normally associated with the Fc region when present in an intact antibody, such as FcRn binding, antibody half life modulation, ADCC function and complement binding.
  • a biologically functional fragment of an antibody is a monovalent antibody that has an in vivo half life substantially similar to an intact antibody.
  • such a biologically functional fragment of an antibody may comprise an antigen binding arm linked to an Fc sequence capable of conferring in vivo stability to the fragment.
  • thioredoxin inhibitor and “Trx inhibitor” are used interchangeably, and include all agents and measures effective in inhibiting thioredoxin activity.
  • thioredoxin (Trx) inhibitors include all agents and measures blocking any component of the Trx, G6PD and/or hexokinase enzyme systems.
  • inhibitor includes complete elimination (blocking) and reduction of thioredoxin activity, and, consequently, complete or partial elimination of disulfide bond reduction in a protein, such as an antibody.
  • an “isolated” antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example, Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • Protein A and “ProA” are used interchangeably herein and encompasses Protein A recovered from a native source thereof, Protein A produced synthetically (e.g. by peptide synthesis or by recombinant techniques), and variants thereof which retain the ability to bind proteins which have a C H 2/C H 3 region, such as an Fc region.
  • Protein A can be purchased commercially from Repligen, GE Healthcare and Fermatech. Protein A is generally immobilized on a solid phase support material.
  • the term “ProA” also refers to an affinity chromatography resin or column containing chromatographic solid support matrix to which is covalently attached Protein A.
  • chromatography refers to the process by which a solute of interest in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.
  • affinity chromatography and “protein affinity chromatography” are used interchangeably herein and refer to a protein separation technique in which a protein of interest or antibody of interest is reversibly and specifically bound to a biospecific ligand.
  • the biospecific ligand is covalently attached to a chromatographic solid phase material and is accessible to the protein of interest in solution as the solution contacts the chromatographic solid phase material.
  • the protein of interest e.g., antibody, enzyme, or receptor protein
  • Binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to be passed 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 removed in active form from the immobilized ligand with low pH, high pH, high salt, competing ligand, and the like, and passed through the chromatographic column with the elution buffer, free of the contaminating proteins or protein impurities that were earlier allowed to pass through the column.
  • Any component can be used as a ligand for purifying its respective specific binding protein, e.g. antibody.
  • 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. antibody) and a solid phase matrix.
  • a “cation exchange resin” refers to a solid phase which is negatively charged, and which thus has free cations for exchange with cations in an aqueous solution passed over or through the solid phase.
  • a negatively charged ligand attached to the solid phase to form the cation exchange resin may, e.g., be a carboxylate or sulfonate.
  • Commercially available cation exchange resins include carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g. SP-SEPHAROSE FAST FLOWTM or SP-SEPHAROSE HIGH PERFORMANCETM, from GE Healthcare) and sulphonyl immobilized on agarose (e.g.
  • a “mixed mode ion exchange resin” refers to a solid phase which is covalently modified with cationic, anionic, and hydrophobic moieties.
  • a commercially available mixed mode ion exchange resin is BAKERBOND ABXTM (J. T. Baker, Phillipsburg, N.J.) containing weak cation exchange groups, a low concentration of anion exchange groups, and hydrophobic ligands attached to a silica gel solid phase support matrix.
  • anion exchange resin is used herein to refer to a solid phase which is positively charged, e.g. having one or more positively charged ligands, such as quaternary amino groups, attached thereto.
  • commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEXTM and FAST Q SEPHAROSETM (GE Healthcare).
  • a “buffer” is a solution that resists changes in pH by the action of its acid-base conjugate components.
  • Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems , Gueffroy, D., ed. Calbiochem Corporation (1975).
  • the buffer has a pH in the range from about 2 to about 9, alternatively from about 3 to about 8, alternatively from about 4 to about 7 alternatively from about 5 to about 7.
  • Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.
  • the “loading buffer” is that which is used to load the composition comprising the polypeptide molecule of interest and one or more impurities onto the 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) is/are bound to the ion exchange resin or such that the protein of interest flows through the column while the impurities bind to the resin.
  • the “intermediate buffer” is used to elute one or more impurities from the ion exchange resin, prior to eluting the polypeptide molecule of interest.
  • the conductivity and/or pH of the intermediate buffer is/are such that one or more impurity is eluted from the ion exchange resin, but not significant amounts of the polypeptide of interest.
  • wash buffer when used herein refers to a buffer used to wash or re-equilibrate the ion exchange resin, prior to eluting the polypeptide molecule of interest. Conveniently, the wash buffer and loading buffer may be the same, but this is not required.
  • 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/are such that the polypeptide of interest is eluted from the ion exchange resin.
  • a “regeneration buffer” may be used to regenerate the ion exchange resin such that it can be re-used.
  • the regeneration buffer has a conductivity and/or pH as required to remove substantially all impurities and the polypeptide of interest from the ion exchange resin.
  • substantially similar denotes a sufficiently high degree of similarity between two numeric values (for example, one associated with an antibody of the invention and the other associated with a reference/comparator antibody), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values).
  • the difference between said two 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% as a function of the reference/comparator value.
  • the difference between said 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 for the reference/comparator molecule.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
  • phage vector refers to a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • viral vector capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector.
  • 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 with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 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 do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • Percent (%) nucleic acid sequence identity is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a reference Factor D-encoding sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Sequence identity is then calculated relative to the longer sequence, i.e. even if a shorter sequence shows 100% sequence identity with a portion of a longer sequence, the overall sequence identity will be less than 100%.
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. “Treatment” herein encompasses alleviation of the disease and of the signs and symptoms of the particular disease.
  • a “disorder” is any condition that would benefit from treatment with the protein. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
  • disorders to be treated herein include carcinomas and allergies.
  • “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, non-human higher primates, other vertebrates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.
  • the mammal is human.
  • Interfering RNA or “small interfering RNA (siRNA)” is a double stranded RNA molecule less than about 30 nucleotides in length that reduces expression of a target gene. Interfering RNAs may be identified and synthesized using known methods (Shi Y., Trends in Genetics 19(1):9-12 (2003), WO/2003056012 and WO2003064621), and siRNA libraries are commercially available, for example from Dharmacon, Lafayette, Colo. Frequently, siRNAs can be successfully designed to target the 5′ end of a gene.
  • the present invention concerns methods for the prevention of the reduction of disulfide bonds of proteins during recombinant production.
  • the invention concerns methods for preventing the reduction of disulfide bonds of recombinant proteins during processing following fermentation.
  • the methods of the invention are particularly valuable for large scale production of disulfide bond containing proteins, such as at a manufacturing scale.
  • the methods of the invention are useful for large scale protein production at a scale of greater than 5,000 L.
  • disulfide bond reduction occurs during processing of the Harvested Cell Culture Fluid (HCCF) produced during manufacturing of recombinant proteins that contain disulfide bonds.
  • HCCF Harvested Cell Culture Fluid
  • this reduction is observed after cell lysis, especially mechanical cell lysis during harvest operations, when it reaches a certain threshold, such as, for example, from about 30% to about 70%, or from about 40% to about 60%, or from about 50% to about 60% total cell lysis.
  • This threshold will vary, depending on the nature of the protein (e.g. antibody) produced, the recombinant host, the production system, production parameters used, and the like, and can be readily determined experimentally.
  • Such reduction might result from a variety of factors and conditions during the manufacturing process, and might be caused by 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 an active thioredoxin (Trx) or thioredoxin-like system in the HCCF.
  • Trx The Trx enzyme system, composed of Trx, thioredoxin reductase (TrxR) and NADPH, is a hydrogen donor system for reduction of disulfide bonds in proteins.
  • Trx is a small monomeric protein with a CXXC active site motif that catalyzes many redox reactions through thiol-disulfide exchange.
  • the oxidized Trx can be reduced by NADPH via TrxR.
  • the reduced Trx is then able to catalyze the reduction of disulfides in proteins.
  • the NADPH required for thioredoxin system is provided via reactions in pentose phosphate pathway and glycolysis.
  • NADPH which is required for activity of the Trx system is provided by glucose-6-phosphate dehyrogenase (G6PD) activity, which generates NADPH from glucose and ATP by hexokinase (see FIG. 4 ).
  • G6PD glucose-6-phosphate dehyrogenase
  • These cellular enzymes (Trx system, G6PD, and hexokinase) along with their substrates are released into the CCF upon cell lysis, allowing reduction to occur. Accordingly, disulfide reduction can be prevented by inhibitors of the Trx enzyme system or upstream enzyme systems providing components for an active Trx system, such as G6PD and hexokinase activity.
  • 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.”
  • the Trx inhibitors are typically added to the cell culture fluid (CCF), which contains the recombinant host cells and the culture media, and/or to the harvested cell culture fluid (HCCF), which is obtained after harvesting by centrifugation, filtration, or similar separation methods.
  • the HCCF lacks intact host cells but typically contains host cell proteins and other contaminants, including DNA, which are removed in subsequent purification steps.
  • the Trx inhibitors may be added before harvest and/or during harvest, preferably before harvest.
  • non-specific methods can also be used to prevent the reduction of disulfide bond reduction following fermentation during the recombinant production of recombinant proteins, such as air sparging or pH adjustment.
  • Certain reduction inhibition methods contemplated herein are listed in the following Table 1.
  • Trx inhibitors for use in the methods of the present invention include, without limitation, (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., the 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 form readily complexe
  • Mg 2+ such as EDTA, and compounds that react with SH groups, sorbose-1-phosphate, polyphosphates, 6-deoxy-6-fluoroglucose, 2-C-hydroxy-methylglucose, xylose and lyxose (Sols et al., 1958, supra; McDonald, 1955, supra); further hexokinase inhibitors are disclosed in U.S. Pat. No. 5,854,067 entitled “Hexokinase Inhibitors.” It will be understood that these inhibitors are listed for illustration only. Other Trx inhibitors exists and can be used, alone or in various combinations, in the methods of the present invention.
  • Trx inhibitors for use in the methods of the present invention also include reagents whereby the reduction of recombinantly produced antibodies or proteins may be reduced or prevented by decreasing the levels of enzymes of the Trx system, the pentose phosphate pathway or hexokinase at various points during the production campaign. In some embodiments, this reduction of enzyme levels may be accomplished by the use of targeted siRNAs, antisense nucleotides, or antibodies. To design targeted siRNAs or antisense nucleotides to the genes as found in CHO cells, these gene sequences are available from public databases to select sequences for targeting enzymes in different organisms. See Example 9 below for examples of the genes of the E. coli and mouse Trx system.
  • a recombinant protein in addition to using inhibitors discussed above, it is also possible in certain embodiments of the instant invention to prevent the reduction of a recombinant protein to be purified by sparging the HCCF with air to maintain an oxidizing redox potential in the HCCF.
  • This is a non-directed measure that can deplete glucose, G6P and NADPH by continuously oxidizing the reduced forms of Trx and TrxR.
  • Air sparging of the HCCF tank can be performed, for example, with an air flow of about 100 liters to about 200 liters, such as, for example, 150 liters per minutes.
  • Air sparging can be performed to reach an endpoint percentage of saturation; for example, air sparging can be continued until the HCCF is about 100% saturated with air, or it can be continued until the HCCR is about 30% saturated with air, or until it is between about 100% saturated to about 30% saturated with air.
  • the minimum amount of dissolved oxygen (dO 2 ) required for the desired inhibitory effect also depends on the antibody or other recombinant protein produced. Thus, for example, about 10% dO 2 (or about 10 sccm for continuous stream) will have the desired effect during the production of antibody 2H7 (Variant A), while Apomab might require a higher (about 30%) dO 2 .
  • another non-directed method usable to block the reduction of the recombinant protein is lowering the pH of the HCCF.
  • This embodiment takes advantage of 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 is significantly lower at pH values below 6, and thus the reduction of the recombinant protein, such as ocrelizumab, can be inhibited.
  • the non-directed approaches can also be combined with each other and/or with the use of one or more Trx inhibitors.
  • Disulfide bond reduction can be inhibited (i.e., partially or fully blocked) by using one or more Trx inhibitors and/or applying non-directed approaches following completion of the cell culture process, preferably to CCF prior to harvest or in the HCCF immediately after harvest.
  • Trx inhibitors preferably to CCF prior to harvest or in the HCCF immediately after harvest.
  • the optimal time and mode of application and effective amounts depend on the nature of the protein to be purified, the recombinant host cells, and the specific production method used. Determination of the optimal parameters is well within the skill of those of ordinary skill in the art.
  • cupric sulfate in the form of pentahydrate or the anhydrous form
  • CCF or HCCF in the concentration range of from about 5 ⁇ M to about 100 ⁇ M, such as from about 10 ⁇ M to about 80 ⁇ M, preferably from about 15 ⁇ M to about 50 ⁇ M.
  • copper e.g. about 0.04 ⁇ M CuSO 4 for the CHO cell cultures used in the Examples herein
  • this amount is in addition to the copper, if any, already present in the cell culture.
  • Any copper (II) salt can be used instead of CuSO 4 as long as solubility is not an issue.
  • copper acetate and copper chloride which are both soluble in water, can be used instead of CuSO 4 .
  • the minimum effective concentration may also depend on the antibody produced and the stage where the inhibitor is used.
  • EDTA can be used in a wide concentration range, depending on the extent of cell lysis, the recombinant host cell used, and other parameters of the production process.
  • EDTA can be typically added in a concentration of between about 5 mM to about 60 mM, such as from about 10 mM to about 50 mM, or from about 20 mM to about 40 mM, depending on the extent of cell lysis.
  • concentrations of EDTA will suffice, while for a cell lysis of about 75%-100%, the required EDTA concentration is higher, such as, for example, from about 20 mM to about 40 mM.
  • the minimum effective concentration may also depend on the antibody produced. Thus, for example, for antibody 2H7 (Variant A) the minimum effective EDTA concentration is about 10 mM.
  • DHEA as a Trx inhibitor is typically effective at a lower concentration, such as for example, in the concentration range from about 0.05 mM to about 5 mM, preferably from about 0.1 mM to about 2.5 mM.
  • Trx inhibitors such as aurothioglucose (ATG) and aurothiomalate (ATM) inhibit reduction of disulfide bonds in the ⁇ M concentration range.
  • ATG or ATM may be added in a concentration between about 0.1 mM to about 1 mM. While the minimum inhibitory concentration varies depending on the actual conditions, for ATG and ATM typically it is around 4 ⁇ TrxR concentration.
  • the mammalian host cell used in the manufacturing process is a chinese hamster ovary (CHO) cell (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)).
  • Other mammalian host cells include, without limitation, monkey kidney CV1 line transformed by 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.
  • 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.
  • MRC 5 cells MRC 5 cells; FS4 cells; a 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).
  • Hep G2 human hepatoma line
  • myeloma or lymphoma cells e.g. Y0, J558L, P3 and NS0 cells
  • a preferred host cell for the production of the polypeptides herein is the CHO cell line DP12 (CHO K1 dhfr ⁇ ). This is one of the best known CHO cell lines, widely used in laboratory practice (see, for example, EP 0,307,247, published Mar. 15, 1989). In addition, other CHO-K1 (dhfr ⁇ ) cell lines are known and can be used in the methods of the present invention.
  • the mammalian host cells used to produce peptides, polypeptides and proteins can be cultured in a variety of media.
  • Commercially available 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 the host cells.
  • any of these media may be supplemented as necessary 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), nucleosides (such as adenosine and thymidine), antibiotics (such as GentamycinTM drug), 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 would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • a protocol for the production, recovery and purification of recombinant antibodies in mammalian, such as CHO, cells may include the following steps:
  • Cells may be cultured in a stirred tank bioreactor system and a fed batch culture, procedure is employed.
  • a fed batch culture the mammalian host cells and culture medium are supplied to a culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.
  • the fed batch culture can include, for example, a semi-continuous fed batch culture, wherein periodically whole culture (including cells and medium) is removed and replaced by fresh medium.
  • Fed batch culture is distinguished from simple batch culture in which all components for cell culturing (including the cells and all culture nutrients) are supplied to the culturing vessel at the start of the culturing process.
  • Fed batch culture can be further distinguished from perfusion culturing insofar as the supernate is not removed from the culturing vessel during the process (in perfusion culturing, the cells are restrained in the culture by, e.g., filtration, encapsulation, anchoring to microcarriers etc. and the culture medium is continuously or intermittently introduced and removed from the culturing vessel).
  • the cells of the culture may be propagated according to any scheme or routine that may be suitable for the particular host cell and the particular production plan contemplated. Therefore, a single step or multiple step culture procedure may be employed.
  • a single step culture the host cells are inoculated into a culture environment and the processes are employed during a single production phase of the cell culture.
  • a multi-stage culture can be used.
  • cells may be cultivated in a number of steps or phases. For instance, cells may be grown in a first step or growth phase culture wherein cells, possibly removed from storage, are inoculated into a medium suitable for promoting growth and high viability. The cells may be maintained in the growth phase for a suitable period of time by the addition of fresh medium to the host cell culture.
  • fed batch or continuous cell culture conditions may be devised to enhance growth of the mammalian cells in the growth phase of the cell culture.
  • cells are grown under conditions and for a period of time that is maximized for growth.
  • Culture conditions such as temperature, pH, dissolved oxygen (dO 2 ) and the like, are those used with the particular host and will be apparent to the ordinarily skilled artisan.
  • the pH is adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., CO 2 ) or a base (e.g., Na 2 CO 3 or NaOH).
  • a suitable temperature range for culturing mammalian cells such as CHO cells is between about 30° C. to 38° C., and a suitable dO 2 is between 5-90% of air saturation.
  • the cells may be used to inoculate a production phase or step of the cell culture.
  • the production phase or step may be continuous with the inoculation or growth phase or step.
  • the cell culture environment during the production phase of the cell culture is typically controlled.
  • factors affecting cell specific productivity of the mammalian host cell may be manipulated such that the desired sialic acid content is achieved in the resulting glycoprotein.
  • the production phase of the cell culture process is preceded by a transition phase of the cell culture in which parameters for the production phase of the cell culture are engaged. Further details of this process are found in U.S. Pat. No. 5,721,121, and Chaderjian et al., Biotechnol. Prog. 21(2):550-3 (2005), the entire disclosures of which are expressly incorporated by reference herein.
  • the first step of a purification process involves lysis of the cell, which can be done by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. The same problem arises, although on a smaller scale, with directly secreted proteins due to the natural death of cells and release of intracellular host cell proteins and components in the course of the protein production run.
  • the desired protein is separated from impurities when the impurities specifically adhere to the column, and the protein of interest does not, that is, the protein of interest is present in the “flow-through.”
  • purification of recombinant proteins from the cell culture of mammalian host cells may include one or more affinity (e.g. protein A) and/or ion exchange chomarographic steps.
  • Ion exchange chromatography is a chromatographic technique that is commonly used for the purification of proteins.
  • ion exchange chromatography charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided 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 and thereby altering the charge of the solute is another way to achieve elution of the solute.
  • the change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution). In the past, these changes have been progressive; i.e., the pH or conductivity is increased or decreased in a single direction.
  • yeast host cells such as common baker's yeast or Saccharomyces cerevisiae
  • suitable vectors include episomally-replicating vectors based on the 2-micron plasmid, integration vectors, and yeast artificial chromosome (YAC) vectors.
  • yeast 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.
  • 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., Proc. Natl. Acad. Sci. USA, 81: 1470 1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4: 475 479 (1985)).
  • Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis , and Rhodotorula .
  • yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis , and Rhodotorula .
  • a list of specific species that are exemplary of this class of yeasts may 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.
  • suitable vectors include baculoviral vectors.
  • suitable expression vectors include vectors derived from the Ti plasmid of Agrobacterium tumefaciens.
  • Prokaryotic host cells suitable for expressing antibodies and other proteins to be protected by means of the instant invention include Archaebacteria and Eubacteria , such as Gram-negative or Gram-positive organisms.
  • useful bacteria include Escherichia (e.g., E. coli ), Bacilli (e.g., B. subtilis ), Enterobacteria, Pseudomonas species (e.g., P. aeruginosa ), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla , or Paracoccus .
  • E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, including strain 33D3 having genotype W3110 ⁇ fhuA ( ⁇ tonA) ptr3 lac Iq lacL8 ⁇ ompT ⁇ (nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635).
  • Other strains and derivatives thereof such as E. coli 294 (ATCC 31,446), E. coli B, E.
  • coli, Serratia , or Salmonella species can be suitably used as the host when well known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
  • the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may desirably be incorporated in the cell culture.
  • Cell lysis is typically accomplished using mechanical disruption techniques such as homogenization or head milling. While the protein of interest is generally effectively liberated, such techniques have several disadvantages (Engler, Protein Purification Process Engineering , Harrison eds., 37 55 (1994)). Temperature increases, which often occur during processing, may result in inactivation of the protein. Moreover, the resulting suspension contains a broad spectrum of contaminating proteins, nucleic acids, and polysaccharides. Nucleic acids and polysaccharides increase solution viscosity, potentially complicating subsequent processing by centrifugation, cross-flow filtration, or chromatography. Complex associations of these contaminants with the protein of interest can complicate the purification process and result in unacceptably low yields. Improved methods for purification of heterologous polypeptides from microbial fermentation broth or homogenate are described, for example, in U.S. Pat. No. 7,169,908, the entire disclosure of which is expressly incorporated herein by reference.
  • the methods of the present invention are used to prevent the reduction of inter- 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 (HERCEPTIN®) (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” as in U.S. Pat. No. 5,736,137 (RITUXAN®), a chimeric or humanized variant of the 2H7 antibody as in U.S. Pat. No.
  • anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN® (Kim et al., Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331, published Oct.
  • 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., J. Immunol. 151:2623-2632 (1993), and International Publication No. WO 95/19181
  • anti-CD18 U.S. Pat. No. 5,622,700, issued Apr.
  • anti-IgE including E25, E26 and E27; U.S. Pat. No. 5,714,338, issued Feb. 3, 1998 or U.S. Pat. No. 5,091,313, issued Feb. 25, 1992, WO 93/04173 published Mar. 4, 1993, or International Application No. PCT/US98/13410 filed Jun. 30, 1998, U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998); anti-TNF- ⁇ antibodies including cA2 (REMICADE®), CDP571 and MAK-195 (See, U.S. Pat. No. 5,672,347 issued Sep.
  • anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and (ZENAPAX®) (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); anti-CD4 antibodies such as the 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 the M22 antibody directed against Fc ⁇ RI as in Graziano et al., J. Immunol.
  • anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al., Cancer Res. 55(23 Suppl): 5935s-5945s (1995); antibodies directed 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(23 Supp): 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al., Eur J. Immunol.
  • anti-CD38 antibodies e.g. AT 13/5 (Ellis et al., J. Immunol. 155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195 (Jurcic et 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 (PANOREX®); anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO®); anti-RSV antibodies such as MEDI-493 (SYNAGIS®); anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such as PRO542; anti-hepatitis antibodies such as the anti
  • the methods of the present invention are used for the production of the following antibodies and recombinant proteins.
  • Rituximab (RITUXAN®) is a genetically engineered chimeric murine/human monoclonal antibody directed against the CD20 antigen.
  • Rituximab is the antibody called “C2B8” in U.S. Pat. No. 5,736,137 issued Apr. 7, 1998 (Anderson et al.).
  • Rituximab is indicated for the treatment of patients with relapsed or refractory low-grade or follicular, CD20-positive, B cell non-Hodgkin's lymphoma.
  • rituximab binds human complement and lyses lymphoid B cell lines through complement-dependent cytotoxicity (CDC) (Reff et al., Blood 83(2):435-445 (1994)). Additionally, it has significant activity in assays for antibody-dependent cellular cytotoxicity (ADCC). More recently, rituximab has been shown to have anti-proliferative effects in tritiated thymidine incorporation assays and to induce apoptosis directly, while other anti-CD19 and CD20 antibodies do not (Maloney et al., Blood 88(10):637a (1996)).
  • rituximab sensitizes drug-resistant human B cell lymphoma cell lines to the cytotoxic effects of doxorubicin, CDDP, VP-1 6, diphtheria toxin and ricin (Demidem et al., Cancer Chemotherapy & Radiopharmaceuticals 12(3):177-186 (1997)).
  • Patents and patent publications concerning CD20 antibodies include U.S. Pat. Nos. 5,776,456, 5,736,137, 6,399,061, and 5,843,439, as well as U.S. patent application Nos. US 2002/0197255A1, US 2003/0021781A1, US 2003/0082172 A1, US 2003/0095963 A1, US 2003/0147885 A1 (Anderson et al.); U.S. Pat. No.
  • the invention provides pharmaceutical compositions comprising humanized 2H7 anti-CD20 antibodies.
  • the humanized 2H7 antibody is an antibody listed in Table 2.
  • Each of the antibody variants A, B and I of Table 2 comprises the light chain variable sequence (V L ):
  • V H the heavy chain variable sequence
  • Each of the antibody variants C, D, F and G of Table 2 comprises the light chain variable sequence (V L ):
  • V H the heavy chain variable sequence
  • the antibody variant H of Table 2 comprises the light chain variable sequence (V L ) of SEQ ID NO:3 (above) and the heavy chain variable sequence (V H ):
  • Each of the antibody variants A, B and I of Table 2 comprises the full length light chain sequence:
  • Variant A of Table 2 comprises the full length heavy chain sequence:
  • Variant B of Table 2 comprises the full length heavy chain sequence:
  • Variant I of Table 2 comprises the full length heavy chain sequence:
  • Each of the antibody variants C, D, F, G and H of Table 2 comprises the full length light chain sequence:
  • Variant C of Table 2 comprises the full length heavy chain sequence:
  • Variant D of Table 2 comprises the full length heavy chain sequence:
  • Variant F of Table 2 comprises the full length heavy chain sequence:
  • Variant G of Table 2 comprises the full length heavy chain sequence:
  • Variant H of Table 2 comprises the full length heavy chain sequence:
  • the humanized 2H7 antibody of the invention further comprises amino acid alterations in the IgG Fc and exhibits increased binding affinity for human FcRn over an antibody having wild-type IgG Fc, 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.
  • Humanized 2H7 antibody compositions of the present invention include compositions of any of the preceding humanized 2H7 antibodies having a Fc region, wherein about 80-100% (and preferably about 90-99%) of the antibody in the composition comprises a mature core carbohydrate structure which lacks fucose, attached to the Fc region of the glycoprotein.
  • Such compositions were demonstrated herein to exhibit a surprising improvement in binding to Fc(RIIIA(F158), which is not as effective as Fc(RIIIA (V158) in interacting with human IgG.
  • Fc(RIIIA (F158) is more common than Fc(RIIIA (V158) in normal, healthy African Americans and Caucasians. See Lehrnbecher et al., Blood 94:4220 (1999).
  • CHO Chinese Hamster Ovary Cells
  • YB2/0 and Lec13 can produce antibodies with 78 to 98% nonfucosylated species.
  • Shinkawa et al., J. Bio. Chem. 278 (5), 3466-347 (2003) reported that antibodies produced in YB2/0 and Lec13 cells, which have less FUT8 activity, show significantly increased ADCC activity in vitro.
  • a bispecific humanized 2H7 antibody encompasses an antibody wherein one arm of the antibody has at least the antigen binding region of the H and/or L chain of a humanized 2H7 antibody of the invention, and the other arm has V region binding specificity for a second antigen.
  • the second antigen is selected from the group consisting of CD3, CD64, CD32A, CD16, NKG2D or other NK activating ligands.
  • a recombinant humanized version of the murine HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumab or HERCEPTIN®; U.S. Pat. No. 5,821,337) is clinically active in patients with HER2-overexpressing metastatic breast cancers that have received extensive prior anti-cancer therapy (Baselga et al., J. Clin. Oncol. 14:737-744 (1996)).
  • Trastuzumab received marketing approval from the Food and Drug Administration (FDA) Sep. 25, 1998 for the treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein.
  • FDA Food and Drug Administration
  • the anti-HER2 antibody comprises the following V L and V H domain sequences:
  • humanized 2C4 version 574 antibody V L (SEQ ID NO:16) DIQMTQSPSSLSASVGDRVTITC KASQDVSIGVA WYQQKPGKAPKLLIYS ASYRYT GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYYIYPYT FGQ GTKVEIK. and humanized 2C4 version 574 antibody V H (SEQ ID NO:17) EVQLVESGGGLVQPGGSLRLSCAAS GFTFTDYTMD WVRQAPGKGLEWVA D VNPNSGGSIYNQRFK GRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARN L GPSFYFDY WGQGTLVTVSS.
  • the anti-HER2 antibody comprises the V L (SEQ ID NO:18) and V H (SEQ ID NO:19) domain sequences of trastuzumab as shown in FIG. 21 and FIG. 22 , respectively.
  • HER2 antibodies with various properties have been 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.
  • anti-VEGF antibodies may, for example, comprise the following sequences:
  • the anti-VEGF antibody comprises the following V L sequence
  • the anti-VEGF antibody comprises the following V L sequence
  • the anti-VEGF antibody comprises the following V L sequence
  • the humanized anti-CD11a antibody efalizumab or Raptiva® received marketing approval from the Food and Drug Administration on Oct. 27, 2003 for the treatment for the treatment of psoriasis.
  • One embodiment provides for an anti-human CD11a antibody comprising the V L and V H sequences of HuMHM24 below:
  • V L DIQMTQSPSSLSASVGDRVTITCRASKTISKYLAWYQQKPGKAPKLLIYS GSTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPLTFGQ GTKVEIKR; and V H (SEQ ID NO:27): EVQLVESGGGLVQPGGSLRLSCAASGYSFTGHWMNWVRQAPGKGLEWVGM IHPSDSETRYNQKFKDRFTISVDKSKNTLYLQMNSLRAEDTAVYYCARGI YFYGTTYFDYWGQGTLVTVSS.
  • the anti-human CD11a antibody may comprise the V H of SEQ ID NO:27 and the full length L chain of HuMHM24 having the sequence of:
  • Antibodies to the DR5 receptor (anti-DR5) antibodies can also be produced in accordance with the present invention.
  • Such anti-DR5 antibodies specifically include all antibody variants disclosed in PCT Publication No. WO 2006/083971, such as the anti-DR5 antibodies designated Apomabs 1.1, 2.1, 3.1, 4.1, 5.1, 5.2, 5.3, 6.1, 6.2, 6.3, 7.1, 7.2, 7.3, 8.1, 8.3, 9.1, 1.2, 2.2, 3.2, 4.2, 5.2, 6.2, 7.2, 8.2, 9.2, 1.3, 2.2, 3.3, 4.3, 5.3, 6.3, 7.3, 8.3, 9.3, and 25.3, especially Apomab 8.3 and Apomab 7.3, preferably Apomab 7.3.
  • the entire content of WO 2006/083971 is hereby expressly incorporated by reference.
  • tissue plasminogen activators such as human tissue plasminogen activator (htPA, alteplase, ACTUVASE®), a thrombolytic agent for the treatment of myocardial infarction
  • htPA tissue plasminogen activator
  • TNKaseTM a ht-PA variant with extended half-life and fibrin specificity for single-bolus administration
  • rhGH recombinant human growth hormone
  • rhGH somatropin, NUTROPIN®, PROTROPIN®
  • DNase I recombinant human deoxyribonuclease I
  • disulfide-containing biologically important proteins include growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitorin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); a
  • the antibodies and other recombinant proteins herein can be produced by well known techniques of recombinant DNA technology.
  • the skilled practitioner could generate antibodies directed against an antigen of interest, e.g., using the techniques described below.
  • the antibody herein is directed against an antigen of interest.
  • the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal.
  • antibodies directed against nonpolypeptide antigens are also contemplated.
  • the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor.
  • Exemplary antigens include those proteins described in section (3) below.
  • Exemplary molecular targets for 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 the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and ⁇ v/ ⁇ 3 integrin including either ⁇ or ⁇ subunits thereof (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 of the other antigens mentioned herein.
  • Antigens to which the antibodies listed above bind are specifically included within the scope herein.
  • Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies.
  • immunogens for transmembrane molecules, such as receptors, fragments of these (e.g. the extracellular domain of a receptor) can be used as the immunogen.
  • transmembrane molecules such as receptors
  • fragments of these e.g. the extracellular domain of a receptor
  • cells expressing the transmembrane molecule can be used as the immunogen.
  • Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule.
  • Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl 2 , or R 1 N ⁇ C ⁇ NR, where R and R 1 are different alkyl groups.
  • a protein that is immunogenic in the species to be immunized e.g., keyhole limpet hemocyanin, serum albumin, bovine thyrog
  • Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 ⁇ g or 5 ⁇ g of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of antigen or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites.
  • Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
  • Conjugates also can be made in recombinant cell culture as protein fusions.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
  • a mouse or other appropriate host animal such as a hamster or macaque monkey
  • lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.
  • lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice , pp. 59-103 (Academic Press, 1986)).
  • the hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA.
  • Human myeloma and mouse-human heteromyeloma cell lines also have been described 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)).
  • Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones may be subcloned by limiting dilution procedures and grown 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.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • immunoglobulin purification procedures such as, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • Protein A chromatography procedure described herein is used.
  • DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies).
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • 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 having specificity for an antigen and another antigen-combining site having specificity for a different antigen.
  • monoclonal antibodies can be isolated from antibody phage libraries generated 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) describe the isolation of murine and human antibodies, respectively, using phage libraries.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993)).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may 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)).
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences.
  • Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
  • Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • J H antibody heavy-chain joining region
  • 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)).
  • antibody fragments Traditionally, these fragments were derived via 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 produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage 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)).
  • F(ab′) 2 fragments can be isolated directly from recombinant host cell culture.
  • Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.
  • the antibody of choice is a single chain Fv fragment (scFv) (see WO 93/16185).
  • Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.
  • bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the C H 3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089).
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies can be prepared using chemical linkage.
  • Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab′) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab′ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • One of the Fab′-TNB derivatives is then reconverted to the Fab′-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab′-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab′ portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • the antibodies can be “linear antibodies” as described in Zapata et al., Protein Eng. 8(10):1057-1062 (1995). Briefly, 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.
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147: 60 (1991).
  • the simplest and most straightforward immunoadhesin design combines the binding domain(s) of the adhesin (e.g. the extracellular domain (ECD) of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain.
  • ECD extracellular domain
  • nucleic acid encoding the binding domain of the adhesin will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible.
  • the encoded chimeric polypeptide will retain at least functionally active hinge, C H 2 and C H 3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the C H 1 of the heavy chain or the corresponding region of the light chain.
  • the precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the immunoadhesin.
  • the adhesin sequence is fused to the N-terminus of the Fc domain of immunoglobulin G 1 (IgG 1 ). It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion.
  • the adhesin amino acid sequence is fused to (a) the hinge region and C H 2 and C H 3 or (b) the C H 1, hinge, C H 2 and C H 3 domains, of an IgG heavy chain.
  • the immunoadhesins are assembled as multimers, and particularly as heterodimers or heterotetramers.
  • these assembled immunoglobulins will have known unit structures.
  • a basic four chain structural unit is the form in which IgG, IgD, and IgE exist.
  • a four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds.
  • IgA globulin, and occasionally IgG globulin may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.
  • each A represents identical or different adhesin amino acid sequences
  • V L is an immunoglobulin light chain variable domain
  • V H is an immunoglobulin heavy chain variable domain
  • C L is an immunoglobulin light chain constant domain
  • C H is an immunoglobulin heavy chain constant domain
  • n is an integer greater than 1;
  • Y designates the residue of a covalent cross-linking agent.
  • the adhesin sequences can be inserted between immunoglobulin heavy chain and light chain sequences, such that an immunoglobulin comprising a chimeric heavy chain is obtained.
  • the adhesin sequences are fused to the 3′ end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the C H 2 domain, or between the C H 2 and C H 3 domains. Similar constructs have been reported by Hoogenboom, et al., Mol. Immunol. 28:1027-1037 (1991).
  • an immunoglobulin light chain might be present either covalently associated to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to the adhesin.
  • DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein.
  • the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs.
  • Immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the adhesin portion in-frame to an immunoglobulin cDNA sequence.
  • fusion to genomic immunoglobulin fragments can also be used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); and Stamenkovic et al., Cell 66:1133-1144 (1991)).
  • the latter type of fusion requires the presence of Ig regulatory sequences for expression.
  • cDNAs encoding IgG heavy-chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques.
  • PCR polymerase chain reaction
  • the cDNAs encoding the “adhesin” and the immunoglobulin parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells.
  • Materials and devices used in the experiments described in the experimental examples include: stainless steel vials (mini-tanks, Flow Components, Dublin, Calif.; short (50 cc) and tall (55 cc)); dialysis tubing (Spectra/Por, 6-8000 MWCO, cat. #132645), 0.22 ⁇ m filter (Millipore Millipak Gamma Gold cat. #MPGL04 GH2); phosphate buffered saline (PBS, EMD, cat. #6506); ethylenediaminetetraacetic acid (EDTA, Sigma, cat. #E4884); ⁇ -nicotinamide adenine dinucleotide phosphate (NADPH, Calbiochem, cat.
  • stainless steel vials mini-tanks, Flow Components, Dublin, Calif.; short (50 cc) and tall (55 cc)
  • dialysis tubing Spectra/Por, 6-8000 MWCO, cat. #132645), 0.22
  • Trx (Sigma, cat. #T8690); TrxR (Sigma, cat. #T9698). All chemicals and reagents were used as received with no further purification.
  • Stock solutions of EDTA 250 mM, pH 7.5), CuSO 4 (10 mM), ATG (30 mM), ATM (30 mM), NADPH (75 mM), G6P (300 mM) were prepared for use in the mini-tank time course studies.
  • 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 the CCF was pulled in through the homogenizer to obtain complete cell lysis (membrane breakage) after a single pass.
  • the CCF homogenate was collected once water was purged through the system.
  • the homogenate was transferred to centrifuge bottles and centrifuged in a Sorval RC-3B rotor centrifuge at 4,500 rpm for 30 minutes at 20° C.
  • centrate was decanted and then depth filtered followed by 0.22 ⁇ m sterile filtration using a peristaltic pump with silicon tubing to generate the final HCCF from the homogenized CCF (100% cell lysis).
  • the CCF was centrifuged straight from the fermentor without any homogenization and then the centrate was filtered with a sterile 0.22 ⁇ m filter to generate the HCCF.
  • a laminar flow hood was used in handling all mini-tanks and all materials used in the HCCF incubation experiments were either autoclaved or rinsed using 70% isopropanol to minimize bacterial contamination.
  • a dialysis experiment was carried out in order to determine whether the components causing reduction of ocrelizumab were small molecules or macromolecules (i.e. enzymes).
  • a sample of 3 mL of purified and formulated ocrelizumab (30.2 mg/mL) was dialyzed against 1 L of phosphate buffered saline (PBS, 10 mM pH 7.2) for 24 hours and the PBS was changed after 8 hours. The concentration of the ocrelizumab sample was then adjusted to 1 mg/mL using the absorbance at 280 nm. Aliquots were stored at ⁇ 70° C. prior to use. Dialysis tubing was hydrated overnight in a 0.05% azide solution and rinsed with sterile water prior to use.
  • PBS phosphate buffered saline
  • the HCCF obtained from homogenization of CCF from a 3-L fermentor was thawed and filtered through a 0.22 ⁇ m Millipak filter using a peristaltic pump.
  • Six short mini-tanks were filled with 30 mL of HCCF each.
  • 500 ⁇ L of ocrelizumab sample in sealed dialysis tubing was added.
  • the mini-tanks were sealed and loaded into a bench top mixer (Barnstead Lab-Line MAX Q 4000) operating at 35 rpm and ambient temperature.
  • one mini-tank was removed from the mixer, and aliquots of the HCCF (in the mini-tank) and ocrelizumab sample (in the dialysis bag) were taken and stored at ⁇ 70° C. until analyzed with the free thiol assay and the Bioanalyzer assay (described below).
  • a tall mini-tank was filled with 27 mL of HCCF.
  • various reagents NADPH, G6P, inhibitors of G6PD or TrxR
  • PBS 10 mM pH 7.2
  • the mini-tanks were sealed and loaded into a bench top mixer running at 35 rpm and ambient temperature.
  • the exteriors of the mini-tanks were sterilized with 70% IPA and opened in a laminar flow hood for the removal of an aliquot.
  • the mini-tanks were then re-sealed and loaded back into the bench top mixer. All aliquots were stored at ⁇ 70° C. until analyzed with the free thiol assay and Bioanalyzer assay (described below).
  • TrxR rat liver
  • lyophilized Trx human was reconstituted with PBS (10 mM, pH 7.2) yielding a 500 ⁇ M solution.
  • a solution of 20 mM NADPH and 10 mM ATG and ATM solutions were prepared in water.
  • a standard curve using GSH was generated in PBS (10 mM, pH 6.0 ⁇ 0.05). From a 110 mM GSH solution, standards were prepared at concentrations of 0, 5.5, 11, 22, 44, 55, 110 and 550 ⁇ M through serial dilution. From an acetonitrile stock solution of mBB (10 mM stored at ⁇ 20° C.), a 100 ⁇ M solution of mBB was prepared in PBS (10 mM, pH 10.0 ⁇ 0.05) and stored away from light.
  • a stock solution of either 250 mM EDTA or 50 mM CUSO 4 was added to the CCF prior to homogenization to evaluate a range of final concentrations to prevent antibody disulfide reduction.
  • these solutions were then mixed with the HCCF generated from the non-homogenized CCF (also containing EDTA or CuSO 4 ) in order to dilute and decrease the total level of cell lysis to below the 100% maximum.
  • a stock solution of 1 M acetic acid was added to a final blended HCCF solution (homogenized CCF and non-homogenized CCF) to decrease the pH of the solution to prevent antibody disulfide reduction.
  • each HCCF solution (containing EDTA, CuSO 4 , acetic acid, or no addition for the control) was held in a 50 mL 316L stainless steel vial.
  • the vial was sealed with a clamp, and the solution was not aerated or agitated.
  • the vial was stored at room temperature (18-22° C.). At predetermined time points, the solution was removed and purified over a lab scale protein A affinity resin.
  • Solutions were either sparged with air to increase the dissolved oxygen level to air saturation or with nitrogen (control) to remove any dissolved oxygen in solution.
  • Gas flow to each vessel was variable dependent upon whether a constant aeration rate was used or a minimum level of dissolved oxygen was maintained.
  • 25-50 mL samples were removed from both vessels and purified over a lab scale protein A affinity resin prior to analysis.
  • Antibody in harvested cell culture fluid samples can be captured and purified using a specific affinity chromatography resin.
  • Protein A resin (Millipore, Prosep-vA High Capacity) was selected as the affinity resin for antibody purification.
  • the resin was packed in a 0.66 cm inner diameter glass column (Omnifit®) with a 14 cm bed height resulting in a 4.8 mL final column volume. Chromatography was performed using an AKTA Explorer 100 chromatography system (GE Healthcare).
  • the resin was exposed to buffers and HCCF at a linear flow rate between 350-560 cm/hr.
  • the resin was equilibrated with 25 mM Tris, 25 mM NaCl, 5 mM EDTA, pH 7.1.
  • the resin was loaded between 5-15 mg antibody per mL of resin.
  • the antibody concentration in the HCCF was determined using an immobilized protein A HPLC column (Applied Biosystems, POROS A). After loading, the resin was washed with 25 mM Tris, 25 mM NaCl, 5 mM EDTA, 0.5 M TMAC, pH 7.1, and then the antibody was eluted using 0.1M acetic acid, pH 2.9.
  • Elution pooling was based on UV absorbance at 280 nm measured inline after the column.
  • the purified elution pools were pH-adjusted using 1 M Sodium HEPES to pH 5.0-5.5. After regeneration of the resin with 0.1 M phosphoric acid, the same or similar packed resins were used for subsequent purification of other HCCF solutions.
  • the antibody concentration in the purified protein A-pool was measured using UV spectrometry at 280 nm.
  • the purified protein A elution pools were analyzed by the Bioanalyzer assay to quantitate the percentage of intact antibody at 150 kDa molecular weight.
  • a dialysis experiment was designed and carried out to determine if the reduction of ocrelizumab was caused by small reducing molecules or macromolecules (e.g., enzymes).
  • purified intact ocrelizumab was placed in a dialysis bag with a molecular weight cut off (MWCO) of 7000 and incubated the dialysis bag in HCCF containing ocrelizumab in a stainless steel mini-tank.
  • MWCO molecular weight cut off
  • Trx system was tested for its ability to reduce ocrelizumab in vitro by incubating intact ocrelizumab with Trx, TrxR, and NADPH.
  • the Bioanalyzer results indicate that ocrelizumab was reduced in vitro by the Trx system ( FIG. 5 ).
  • the rate of reduction in this in vitro system appears to be slower than that in the HCCF (for example when compared to the reduction shown in FIG. 2 ). This is likely due to lower concentrations of the enzymes (Trx and Trx-R) and/or the buffer system used in the in vitro reaction because reaction rate of Trx system is dependent on both the enzyme concentrations and buffer systems.
  • Cupric sulfate is known for its ability to provide oxidizing redox potential and has been used in the cell culture processes to minimize free thiol (i.e., minimize unpaired cysteine) levels in recombinant antibody molecules (Chaderjian et al., 2005, supra). Cupric sulfate was tested for efficacy in inhibiting the Trx system in vitro and the subsequent reduction of ocrelizumab. In this in vitro reduction experiment, the buffer system was changed from PBS to histidine sulfate to avoid the formation of insoluble Cu 3 (PO 4 ) 2 .
  • TrxR aurothioglucose
  • ATM aurothiomalate
  • Trx system was active in the HCCF and reduced ocrelizumab as observed in the manufacturing runs resulting in reduced antibody molecules or in the lab scale experiments, both gold compounds (ATG and ATM) should be able to inhibit the reduction of ocrelizumab in HCCF.
  • FIG. 10 shows that ocrelizumab was readily reduced in an HCCF from homogenized CCT generated from a 3-L fermentor after a period of incubation. However, the ocrelizumab reduction event was completely inhibited when either 1 mM ATG or ATM was added to the HCCF ( FIGS. 11 and 12 ). These results demonstrated that the Trx system is active in the HCCF and is directly responsible for the reduction of ocrelizumab.
  • the reduction of disulfides by the Trx system requires the reducing equivalents from NADPH ( FIG. 4 ).
  • the main cellular metabolic pathway that provides NADPH for all reductive biosynthesis reactions is the pentose phosphate pathway.
  • the enzymes in this pathway must be still active in the HCCF in order to keep the Trx system active.
  • the first step in the pentose phosphate pathway (catalyzed by G6PD) must be active to reduce NADP + to NADPH while converting G6P to 6-phosphogluconolactone.
  • G6P is most likely produced from glucose and adenosine 5′-triphosphate (ATP) by the hexokinase activity in HCCF.
  • ATP adenosine 5′-triphosphate
  • HCCF The reducing activity in the HCCF appeared to be transitory in some cases and may be inhibited over time under certain storage conditions or after multiple freeze/thaw cycles. HCCF that has fully lost reducing activity provided an opportunity to explore the role of NADPH and G6P in the reduction of ocrelizumab by Trx system.
  • the hexokinase catalyzes the transfer of phosphate group from Mg2+-ATP to glucose, a reaction that requires the complexation of Mg2+ with ATP (Hammes & Kochavi, 1962a & 1962b, supra). Since EDTA is a metal ion chelator, especially for Mg2+, it can be an effective inhibitor of hexokinase. The observation that an excess amount of EDTA can effectively block the reduction indicates the involvement of hexokinase (i.e. providing G6P) in the mechanism of ocrelizumab reduction.
  • EDTA blocks the reduction of ocrelizumab by eliminating the hexokinase activity and thereby reducing the G6P level available for G6PD, and subsequently the NADPH level available for the Trx system.
  • EDTA is every effective in blocking the reduction of ocrelizumab in fresh HCCF, it was unable to prevent the reduction of ocerlizumab in the beta run HCCF in which the Trx system activity was lost then reactivated by the addition of G6P.
  • the reduction of ocrelizumab was observed in an HCCF incubation experiment in which 5 mM G6P and 20 mM EDTA (final concentrations) were added to the beta run HCCF that had fully lost reducing activity ( FIG. 18 ).
  • no reduction was seen in the control incubation experiment in which no G6P and EDTA were added.
  • the EDTA used in this manner may therefore inhibit neither the Trx system nor the G6PD, and may function as an inhibitor for hexokinase, which produces the G6P for the G6PD. Without G6P, the Trx system would not be supplied with the necessary NADPH for activity.
  • Dehydroepiandrosterone as well as other similar G6PD inhibitors, effectively blocks G6PD activity (Gordon et al., 1995, supra). G6PD inhibitors also prevent the reduction of an antibody in HCCF, for example, ocrelizumab, by blocking the generation of NADPH. The ability of DHEA to inhibit the reduction of orcelizumab is demonstrated in an HCCF incubation experiment. Adding DHEA to a HCCF prevents antibody reduction.
  • DHEA is typically used in the concentration range from about 0.05 mM to about 5 mM. DHEA is also typically used in the concentration range from about 0.1 mM to about 2.5 mM.
  • HCCFs Four different HCCFs were stored and held in the stainless steel vials. The solutions were similar in the amount of cell lysis, which were generated by diluting HCCF from homogenized CCF with HCCF from non-homogenized CCF. For example, 150 mL of the first lysed solution was mixed with 50 mL of the second solution, respectively.
  • the four HCCF mixtures evaluated in this study contained either: (1) 20 mM EDTA, (2) 30 ⁇ M CuSO 4 , (3) 15 mM acetic acid (pH 5.5), and (4) no chemical inhibitor was added for the control solution.
  • HCCF mixture generated from homogenized CCF was stored and held in two separate 10 L stainless steel fermentors.
  • One vessel was sparged with air while the other vessel was sparged with nitrogen gas.
  • 50 mL samples were removed from each vessel and the antibody was purified using protein A chromatography.
  • Purified protein A elution pools were then analyzed by the Bioanalyzer assay to quantitate the percentage of intact antibody at 150 kDa. The results showed that approximately 85% intact antibody was present in the initial solution ( FIG. 20 ), indicating some early reduction of the antibody disulfide bonds prior to exposure to oxygen (i.e.
  • the design of targeted siRNAs or antisense nucleotides to the genes as found in CHO cells may be done by using publicly available sequences such as those for 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 thioreodoxin 2 (SEQ ID NO:33), mouse thioredoxin reductase 1 (SEQ ID NO:34), and mouse thioredoxin reductase 2 (SEQ ID NO:35).
  • 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 thioreodoxin 2 SEQ ID NO:33
  • mouse thioredoxin 1 The sequence of mouse thioredoxin 1 is:
  • mouse thioreodoxin 2 The sequence of mouse thioreodoxin 2 is:
  • mouse thioredoxin reductase 1 The sequence of mouse thioredoxin reductase 1 is:
  • mouse thioredoxin reductase 2 The sequence of mouse thioredoxin reductase 2 is:
  • TrxR rat liver
  • lyophilized Trx human was reconstituted with PBS (10 mM, pH 7.2) yielding a 500 ⁇ M solution.
  • a solution of 20 mM NADPH and 10 mM ATG and ATM solutions were prepared in water.
  • reaction buffer (10 mM histidine, 10 mM Na2SO4, 137 mM NaCl, 2.5 mM KCl, pH 7.0), 25 ⁇ L NADPH, 16 ⁇ L formulated ocrelizumab solution (30.2 mg/mL) and 5 ⁇ L Trx were gently mixed.
  • the reaction was initiated by the addition of 17.5 ⁇ L TrxR.
  • the reaction was incubated at room temperature for 24 hours. Aliquots of 20 ⁇ L were taken at each sampling time-point and stored at ⁇ 70° C. until analyzed by the Bioanalyzer assay.
  • FIG. 24 shows a digital gel-like image from Bioanalyzer analysis (each lane representing a time point) showing that incubation of intact ocrelizumab (“2H7,” a humanized anti-CD20 antibody, referred to as “Variant A” above) (1 mg/mL) with 0.1 ⁇ M TrxR (rat liver), 5 ⁇ M Trx (human) and 1 mM NADPH in 10 mM histidine sulfate buffer results in the reduction of ocrelizumab in less than one hour.
  • 2H7 a humanized anti-CD20 antibody
  • Aurothioglucose was added to the ocrelizumab mixture described above, at the following concentrations: 1 mM; 0.6 ⁇ M (6:1 ATG:TrxR); 0.4 ⁇ M (4:1 ATG:TrxR); and 0.2 ⁇ M (2:1 ATG:TrxR).
  • aurothioglucose added at concentrations 1 mM, 0.6 ⁇ M, and 0.4 ⁇ M effectively inhibits the reduction of ocrelizumab by the thioredoxin system.
  • aurothioglucose added at a concentration of 0.2 ⁇ M cannot inhibit ocrelizumab reduction after 24 hours.
  • ATM Aurothiomalate
  • CuSO 4 was added to the ocrelizumab mixture described above, at concentrations of 20 ⁇ M (4:1 Cu 2+ :Trx); 10 ⁇ m (2:1 Cu 2+ :Trx); and 5 ⁇ M (1:1 Cu 2+ :Trx). As shown in FIGS. 31-33 , CuSO 4 effectively inhibits thioredoxin-induced reduction of ocrelizumab at concentrations of 20 ⁇ M and 10 ⁇ M ( FIGS. 31 and 32 ), but the 5 ⁇ M concentration is insufficient to result in a complete inhibition of reduction ( FIG. 33 ).
  • Cystamine was added to the ocrelizumab mixture describe above at the following concentrations: 532 ⁇ M (20:1 cystamine:2H7 (Variant A) disulfide); 266 ⁇ M (10:1 cystamine:2H7 (Variant A) disulfide); 133 ⁇ M (5:1 cystamine:2H7 disulfide); and 26.6 ⁇ M (1:1 cystamine:2H7 (Variant A) disulfide). As shown in FIGS.
  • cystamine effectively inhibits thioredoxin-induced reduction of ocrelizumab at concentrations of 532 ⁇ M (20:1 cystamine:2H7 (Variant A) disulfide) and 266 ⁇ M (10:1 cystamine:2H7 (Variant A)) ( FIGS. 34 and 35 ) but the 133 ⁇ M (5:1 cystamine:2H7 (Variant A) disulfide) and 26.6 ⁇ M (1:1 cystamine:2H7 (Variant A) disulfide) concentrations are insufficient to inhibit the reduction of ocrelizumab after 24 hours ( FIGS. 36 and 37 ).
  • Cystine was added to the ocrelizumab mixture described above at a concentration of 2.6 mM. As shown in FIG. 38 , at this concentration cystine effectively inhibits reduction of ocrelizumab by the thioredoxin system. It is noted that the minimum effective concentration of cystine (just as the effective minimum concentration of other inhibitors) depends on the actual circumstances, and might be different for different proteins, such as antibodies, and might vary depending on the timing of addition. Thus, for example, if cystine is added pre-lysis, the minimum effective concentration for antibody 2H7 (Variant A) is about 1.3 mM, for Apomab about 1 mM and for antibody Variant C about 4.5 mM.
  • the minimum effective concentration typically is somewhat higher, and is about 5.2 mM for 2H7 (Variant A), 6 mM for Apomab and 9 mM for antibody Variant C.
  • the minimum effective inhibitory concentration is about 40 ⁇ of the antibody concentration (in ⁇ M).
  • GSSG was added to the ocrelizumab mixture described above at a concentration of 2.6 mM. As shown in FIG. 39 , at this concentration GSSG effectively inhibits reduction of ocrelizumab by the thioredoxin system. It is noted, however, that the minimum effective concentration of oxidize glutathione (just as that of the other inhibitors) depends on the actual circumstances, such as, for example, on the nature of the protein (e.g. antibody) produced and the timing of addition. For example, for antibody 2H7 (Variant A) the minimum effective concentration is about 1.3 mM for addition prior to lysis.
  • FIG. 40 shows a digital gel-like image from Bioanalyzer analysis (each lane representing a time point) showing that incubation of intact ocrelizumab (“2H7,” a humanized anti-CD20 antibody, Variant A) (1 mg/mL) with 10 ⁇ g/mL hexokinase, 50 ⁇ g/mL glucose-6-phosphate dehydrogenase, 5 ⁇ M thioredoxin, 0.1 ⁇ M thoredoxin reductase, 2 mM glucose, 0.6 mM ATP, 2 mM Mg 2+ , and 2 mM NADP in 50 mM histidine sulfate buffered at pH 7.38 results in the reduction of ocrelizumab in about one hour. Addition of 0.1 mM HDEA, a known glucose-6-phosphate dehydrogenase inhibitor does not inhibit the reduction.
US12/217,745 2007-07-09 2008-07-08 Prevention of disulfide bond reduction during recombinant production of polypeptides Abandoned US20090053786A1 (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
US12/217,745 US20090053786A1 (en) 2007-07-09 2008-07-08 Prevention of disulfide bond reduction during recombinant production of polypeptides
US13/354,223 US8574869B2 (en) 2007-07-09 2012-01-19 Prevention of disulfide bond reduction during recombinant production of polypeptides
US14/043,758 US20140128575A1 (en) 2007-07-09 2013-10-01 Prevention of disulfide bond reduction during recombinant production of polypeptides
US15/488,917 US20170313780A1 (en) 2007-07-09 2017-04-17 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/240,592 US10759866B2 (en) 2007-07-09 2019-01-04 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,309 US10808037B1 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,317 US10906986B2 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/087,313 US11639395B2 (en) 2007-07-09 2020-11-02 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/124,314 US11078294B2 (en) 2007-07-09 2020-12-16 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/194,471 US20230383004A1 (en) 2007-07-09 2023-03-31 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/310,448 US20230365704A1 (en) 2007-07-09 2023-05-01 Prevention of disulfide bond reduction during recombinant production of polypeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US94867707P 2007-07-09 2007-07-09
US12/217,745 US20090053786A1 (en) 2007-07-09 2008-07-08 Prevention of disulfide bond reduction during recombinant production of polypeptides

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/354,223 Continuation US8574869B2 (en) 2007-07-09 2012-01-19 Prevention of disulfide bond reduction during recombinant production of polypeptides

Publications (1)

Publication Number Publication Date
US20090053786A1 true US20090053786A1 (en) 2009-02-26

Family

ID=39791340

Family Applications (11)

Application Number Title Priority Date Filing Date
US12/217,745 Abandoned US20090053786A1 (en) 2007-07-09 2008-07-08 Prevention of disulfide bond reduction during recombinant production of polypeptides
US13/354,223 Active US8574869B2 (en) 2007-07-09 2012-01-19 Prevention of disulfide bond reduction during recombinant production of polypeptides
US14/043,758 Abandoned US20140128575A1 (en) 2007-07-09 2013-10-01 Prevention of disulfide bond reduction during recombinant production of polypeptides
US15/488,917 Abandoned US20170313780A1 (en) 2007-07-09 2017-04-17 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/240,592 Active US10759866B2 (en) 2007-07-09 2019-01-04 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,309 Active US10808037B1 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,317 Active US10906986B2 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/087,313 Active 2029-07-30 US11639395B2 (en) 2007-07-09 2020-11-02 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/124,314 Active US11078294B2 (en) 2007-07-09 2020-12-16 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/194,471 Pending US20230383004A1 (en) 2007-07-09 2023-03-31 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/310,448 Pending US20230365704A1 (en) 2007-07-09 2023-05-01 Prevention of disulfide bond reduction during recombinant production of polypeptides

Family Applications After (10)

Application Number Title Priority Date Filing Date
US13/354,223 Active US8574869B2 (en) 2007-07-09 2012-01-19 Prevention of disulfide bond reduction during recombinant production of polypeptides
US14/043,758 Abandoned US20140128575A1 (en) 2007-07-09 2013-10-01 Prevention of disulfide bond reduction during recombinant production of polypeptides
US15/488,917 Abandoned US20170313780A1 (en) 2007-07-09 2017-04-17 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/240,592 Active US10759866B2 (en) 2007-07-09 2019-01-04 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,309 Active US10808037B1 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US16/847,317 Active US10906986B2 (en) 2007-07-09 2020-04-13 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/087,313 Active 2029-07-30 US11639395B2 (en) 2007-07-09 2020-11-02 Prevention of disulfide bond reduction during recombinant production of polypeptides
US17/124,314 Active US11078294B2 (en) 2007-07-09 2020-12-16 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/194,471 Pending US20230383004A1 (en) 2007-07-09 2023-03-31 Prevention of disulfide bond reduction during recombinant production of polypeptides
US18/310,448 Pending US20230365704A1 (en) 2007-07-09 2023-05-01 Prevention of disulfide bond reduction during recombinant production of polypeptides

Country Status (24)

Country Link
US (11) US20090053786A1 (zh)
EP (7) EP2188302B1 (zh)
JP (5) JP5687057B2 (zh)
KR (6) KR101643514B1 (zh)
CN (3) CN112480255A (zh)
AU (2) AU2008275229B2 (zh)
BR (1) BRPI0812603B1 (zh)
CA (2) CA3113365A1 (zh)
DE (1) DE17196350T1 (zh)
DK (2) DK3327026T3 (zh)
ES (4) ES2655474T3 (zh)
FI (1) FI3597659T3 (zh)
HR (3) HRP20230461T3 (zh)
HU (4) HUE037162T2 (zh)
IL (6) IL202934B (zh)
LT (2) LT3327026T (zh)
MX (2) MX354993B (zh)
PL (4) PL3597659T3 (zh)
PT (2) PT3597659T (zh)
SG (3) SG10201406411XA (zh)
SI (4) SI3597659T1 (zh)
TR (1) TR201802088T4 (zh)
TW (4) TWI666217B (zh)
WO (1) WO2009009523A2 (zh)

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090010921A1 (en) * 2003-11-05 2009-01-08 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
US20100135987A1 (en) * 2008-10-20 2010-06-03 Hickman Robert K Isolation and purification of antibodies using protein a affinity chromatography
US20100322931A1 (en) * 2009-06-17 2010-12-23 Harding Fiona A Anti-vegf antibodies and their uses
US20110076273A1 (en) * 2009-09-11 2011-03-31 Genentech, Inc. Highly Concentrated Pharmaceutical Formulations
US20110177095A1 (en) * 2009-12-16 2011-07-21 Abbott Biotherapeutics Corporation Anti-her2 antibodies and their uses
US20120157664A1 (en) * 2009-04-30 2012-06-21 Ablynx N.V. Method for the production of domain antibodies
US8574869B2 (en) 2007-07-09 2013-11-05 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US20140080180A1 (en) * 2012-03-27 2014-03-20 Genentech, Inc. Harvest operations for recombinant proteins
US8906646B2 (en) 2006-09-13 2014-12-09 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
US8911734B2 (en) 2010-12-01 2014-12-16 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75
US8921526B2 (en) 2013-03-14 2014-12-30 Abbvie, Inc. Mutated anti-TNFα antibodies and methods of their use
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9067988B2 (en) 2010-12-01 2015-06-30 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US9078878B2 (en) 2010-12-01 2015-07-14 Alderbio Holdings Llc Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
US9109010B2 (en) 2008-10-20 2015-08-18 Abbvie Inc. Viral inactivation during purification of antibodies cross reference to related applications
US9150645B2 (en) 2012-04-20 2015-10-06 Abbvie, Inc. Cell culture methods to reduce acidic species
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9193787B2 (en) 2012-04-20 2015-11-24 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9206390B2 (en) 2012-09-02 2015-12-08 Abbvie, Inc. Methods to control protein heterogeneity
US9234033B2 (en) 2012-09-02 2016-01-12 Abbvie, Inc. Methods to control protein heterogeneity
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US9539324B2 (en) 2010-12-01 2017-01-10 Alderbio Holdings, Llc Methods of preventing inflammation and treating pain using anti-NGF compositions
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions
WO2017027861A1 (en) 2015-08-13 2017-02-16 Amgen Inc. Charged depth filtration of antigen-binding proteins
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
US9815893B2 (en) 2012-11-30 2017-11-14 Abbvie Biotherapeutics Inc. Anti-VEGF antibodies and their uses
US9884909B2 (en) 2010-12-01 2018-02-06 Alderbio Holdings Llc Anti-NGF compositions and use thereof
US9976163B2 (en) 2012-10-03 2018-05-22 Kyowa Hakko Kirin Co., Ltd Method for preventing reduction of polypeptide by adding amino acid to culture solution
US20190119374A1 (en) * 2017-05-17 2019-04-25 Immunogen, Inc. Compositions and methods for antibody production
CN110831964A (zh) * 2017-05-09 2020-02-21 百时美施贵宝公司 在抗体制造期间控制粉红色形成的方法
CN111164102A (zh) * 2017-10-02 2020-05-15 拜耳医药保健有限公司 用亚硒酸防止细胞培养收获物中的二硫键还原的方法
US10653794B2 (en) * 2015-08-12 2020-05-19 Pfizer Inc. Capped and uncapped antibody cysteines, and their use in antibody-drug conjugation
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US11214610B2 (en) 2010-12-01 2022-01-04 H. Lundbeck A/S High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris
CN114703244A (zh) * 2016-05-10 2022-07-05 豪夫迈·罗氏有限公司 重组产生多肽期间减少三硫键的方法
US11414498B2 (en) 2008-01-30 2022-08-16 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US11760777B2 (en) 2017-04-26 2023-09-19 Bristol-Myers Squibb Company Methods of antibody production that minimize disulfide bond reduction
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder

Families Citing this family (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW201014605A (en) 2008-09-16 2010-04-16 Genentech Inc Methods for treating progressive multiple sclerosis
HUE048980T2 (hu) 2009-08-11 2020-08-28 Hoffmann La Roche Fehérjék elõállítása glutaminmentes sejttenyésztõ táptalajban
WO2011121560A2 (en) * 2010-03-31 2011-10-06 Universite De Geneve Stabilized antibody preparations and uses thereof
CN101974535B (zh) * 2010-11-03 2012-12-19 北京天广实生物技术股份有限公司 新型抗ErbB2人源化抗体MIL12的制备及其应用
CN102911265B (zh) * 2011-08-05 2014-02-05 王革 一种人神经生长因子的重组变异体及其制备方法
US9650411B2 (en) 2012-08-07 2017-05-16 Kyowa Hakko Kirin Co., Ltd. Method of purifying protein
AR095863A1 (es) 2013-04-16 2015-11-18 Genentech Inc Variantes de pertuzumab, su evaluación, método de tratamiento, método de preparación y artículo de fabricación
WO2015009699A1 (en) 2013-07-15 2015-01-22 Ketter Patrick Methods and compositions to prevent or treat bacterial infections
KR20160088930A (ko) * 2013-12-04 2016-07-26 이뮤노젠 아이엔씨 항체 생산용 조성물 및 항체 생성 방법
US9620278B2 (en) * 2014-02-19 2017-04-11 General Electric Company System and method for reducing partial discharge in high voltage planar transformers
US10647777B2 (en) * 2014-05-28 2020-05-12 Genzyme Corporation Methods of controlling the formation of disulfide bonds in protein solutions
AR103172A1 (es) * 2014-12-22 2017-04-19 Novartis Ag Reducción selectiva de residuos de cisteina en anticuerpos il-17
US10202435B2 (en) 2016-04-15 2019-02-12 Alder Biopharmaceuticals, Inc. Anti-PACAP antibodies and uses thereof
CN109414491A (zh) * 2016-05-10 2019-03-01 免疫医疗有限责任公司 预防蛋白质二硫键还原
KR102499718B1 (ko) * 2016-06-10 2023-02-13 론자 리미티드 단백질을 안정화시키는 방법
WO2018002036A1 (en) 2016-06-28 2018-01-04 Zaklady Farmaceutyczne Polpharma Sa Recombinant production of monoclonal antibodies
CN106366160B (zh) * 2016-10-11 2019-06-14 厦门大学 基于二硫键精准配对构建富含二硫键多肽分子骨架的方法
US20190374639A1 (en) 2016-11-21 2019-12-12 Polpharma Biologics S.A. Aqueous pharmaceutical formulations
EP3548071A4 (en) 2016-11-30 2020-07-15 OncoMed Pharmaceuticals, Inc. METHOD FOR TREATING CANCER WITH TIGIT-BINDING ACTIVE SUBSTANCES
US20190316096A1 (en) 2016-12-28 2019-10-17 Kyowa Hakko Kirin Co Method for selecting cell
US11697670B2 (en) * 2017-08-22 2023-07-11 Biogen Ma Inc. Methods for purifying antibodies having reduced high molecular weight aggregates
TW201927335A (zh) * 2017-10-02 2019-07-16 美商拜耳保健有限責任公司 防止雙硫鍵於細胞培養收集物中還原之方法
US11447547B1 (en) 2017-12-13 2022-09-20 Amgen Inc. Method of antigen-binding protein production
WO2019213527A1 (en) * 2018-05-04 2019-11-07 Sigma-Aldrich Co. Llc Engineered cells with modified host cell protein profiles
WO2020028616A1 (en) * 2018-08-02 2020-02-06 Lonza Ltd Methods for manufacturing recombinant protein comprising a disulfide bond
CA3117992A1 (en) 2018-11-08 2020-05-14 Sutro Biopharma, Inc. E coli strains having an oxidative cytoplasm
EP3947632A1 (en) 2019-04-01 2022-02-09 The Automation Partnership (Cambridge) Ltd. Operation process for a cell cultivation system
JP2022542863A (ja) 2019-07-24 2022-10-07 ハー・ルンドベック・アクチエゼルスカベット 抗ｍＧｌｕＲ５抗体及びその使用
CN113480652B (zh) * 2021-07-30 2023-04-07 成都景泽生物制药有限公司 一种重组cho细胞发酵培养生产重组egfr抗体活性分子的方法
WO2023049687A1 (en) 2021-09-21 2023-03-30 Bristol-Myers Squibb Company Methods of controlling the level of dissolved oxygen (do) in a solution comprising a recombinant protein in a storage container
US20240092870A1 (en) * 2022-09-15 2024-03-21 Sanofi Methods of determining protein reduction susceptability

Citations (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US524474A (en) * 1894-08-14 Seed-planter
US670534A (en) * 1900-07-27 1901-03-26 Oliver L Carmical Seeding-machine and fertilizer-distributer.
US1369649A (en) * 1920-01-02 1921-02-22 George A Gieseler Pneumatic conveying mechanism
US2062295A (en) * 1932-11-22 1936-12-01 Niagara Sprayer And Chemical C Crop duster
US2171205A (en) * 1937-08-26 1939-08-29 Louis A Zinke Powder distributing attachment for tractors
US2193738A (en) * 1938-05-21 1940-03-12 Nat Conveyors Co Inc Pneumatic conveyer
US2559183A (en) * 1946-12-10 1951-07-03 Lawton L Barnett Transplanter drill set
US2671690A (en) * 1948-02-21 1954-03-09 Ehrenkrook Karl Von Forced air sprayer and duster
US2814531A (en) * 1956-04-27 1957-11-26 Clough Equipment Co Balanced pneumatic conveying systems for pulverulent material
US2937049A (en) * 1949-10-18 1960-05-17 Osawa Hirosaburo Method of and an apparatus for carrying coals out of a vertical shaft with the aid of heavy liquid in the coal mine
US3197261A (en) * 1964-03-09 1965-07-27 Simpson Herbert Corp System for feeding and transporting materials
US3207560A (en) * 1962-12-26 1965-09-21 Monsanto Co Unloading device
US3386474A (en) * 1965-05-26 1968-06-04 Chemagro Corp Diverter valve for finely divided solids
US3387895A (en) * 1966-12-29 1968-06-11 Combustion Eng Non-clogging splitter unit for dividing the flow of fluid-conveyed material
US3515315A (en) * 1966-01-24 1970-06-02 Kidd Designs Ltd Archie Combined seed and fertilizer drills for agricultural use
US3543704A (en) * 1968-08-05 1970-12-01 Deere & Co Seed planter
US3548765A (en) * 1967-12-27 1970-12-22 Noget Gougis Sa Agricultural distributing implement
US4036408A (en) * 1975-12-24 1977-07-19 Acf Industries, Incorporated Hopper having means for directing blasts of gas to a discharge outlet or the hopper
US4082364A (en) * 1974-08-27 1978-04-04 Waeschle Maschinenfabrik Gmbh Method and apparatus for charging bulk material to a plurality of receiving stations
US4200412A (en) * 1978-04-20 1980-04-29 Dynamic Air Incorporated Control for pneumatic conveying system
US4244522A (en) * 1979-08-31 1981-01-13 Deere & Company Liquid storage and distribution apparatus for agricultural implements
US4264243A (en) * 1979-01-04 1981-04-28 Dundee Cement Company Constant vacuum barge unloading system
US4280419A (en) * 1977-03-21 1981-07-28 International Harvester Company Pneumatic system for conveying granular material
US4379664A (en) * 1980-07-11 1983-04-12 Prasco Super Seeder Ltd. Seed cup assembly
US4413935A (en) * 1981-06-29 1983-11-08 Combustion Engineering, Inc. Flow splitter for dividing a stream of pulverulent material into multiple streams
US4506704A (en) * 1981-12-30 1985-03-26 Shell Oil Company Distributing valve
US4553882A (en) * 1982-10-28 1985-11-19 Trutzschler Gmbh & Co. Kg Method and apparatus for pneumatically conveying fiber material
US4562779A (en) * 1984-01-26 1986-01-07 Briggs Manufacturing, Inc. Foldable drill implement
US4674922A (en) * 1984-06-02 1987-06-23 Bernd Federhen Apparatus and process for controlling a charging operation for a feed of particles of bulk material
US4738770A (en) * 1985-11-25 1988-04-19 Cities Service Oil And Gas Corp. Apparatus and method of removal of catalyst blockage from reactor recycle pumps
US4779765A (en) * 1987-04-13 1988-10-25 Deere & Company Fertilizer divider for dual air system
US4793743A (en) * 1987-04-29 1988-12-27 Morris Rod-Weeder Company Ltd. Air seeder purging system
US4843983A (en) * 1988-02-02 1989-07-04 Deere & Company Pneumatic manifold quick coupling
US4872785A (en) * 1988-01-22 1989-10-10 Schrage Timothy L Shut-off means for air-actuated planter
US5033914A (en) * 1989-09-29 1991-07-23 Cyclonaire Corporation High efficiency feeder apparatus for pneumatic conveying lines
US5069583A (en) * 1990-10-26 1991-12-03 Caldwell Lawrence G Device for changing flow paths in a pneumatic conveying system
US5156102A (en) * 1988-11-16 1992-10-20 Kongskilde Maskinfabrik A/S Apparatus for sowing granular material
US5161473A (en) * 1991-05-17 1992-11-10 Deere & Company Seed distribution system and method for a seeding implement
US5240355A (en) * 1992-05-22 1993-08-31 Nol-Tec Systems, Inc. Dense phase transporter pneumatic conveying system
US5379706A (en) * 1993-04-07 1995-01-10 Agco Corporation Seed distribution system for planters and drills
US5392722A (en) * 1993-04-20 1995-02-28 Deere & Company Air separation tube for a pneumatic delivery system
US5407305A (en) * 1993-04-29 1995-04-18 Semco, Inc. Continuous dense phase conveying method utilizing high pressure gas at predetermined gas pressures within a conveying pipe
US5494381A (en) * 1991-04-11 1996-02-27 The Young Industries, Inc. Apparatus and method for pneumatically conveying bulk materials
US5575225A (en) * 1995-02-27 1996-11-19 Unverferth Manufacturing Company, Inc. Air-powered apparatus for filling grain drills and method of using same
US5749682A (en) * 1996-09-10 1998-05-12 Renfro Corporation Article collection and distribution system
US5813801A (en) * 1996-06-28 1998-09-29 Mac Equipment, Inc. Dense phase particulate conveying system and method with continuous air leakage management
US5878679A (en) * 1997-08-18 1999-03-09 Deere & Company Product disconnect for metering device
US5927217A (en) * 1997-06-16 1999-07-27 Conserva Pak Seeding Systems Metering particulate material
US6047652A (en) * 1998-05-12 2000-04-11 Case Corporation Seed planter distribution system
US6253693B1 (en) * 1998-11-05 2001-07-03 Flexi-Coil Ltd. Nurse adapter for air seeders

Family Cites Families (221)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3845702A (en) 1970-04-01 1974-11-05 Mies Filter Prod Inc Apparatus for cooking chicken
US4049494A (en) 1975-09-04 1977-09-20 The United States Of America As Represented By The Secretary Of Agriculture Vaccine production process
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
FR2413974A1 (fr) 1978-01-06 1979-08-03 David Bernard Sechoir pour feuilles imprimees par serigraphie
US4515893A (en) 1979-04-26 1985-05-07 Ortho Pharmaceutical Corporation Hybrid cell line for producing complement-fixing monoclonal antibody to human T cells
US4943529A (en) 1982-05-19 1990-07-24 Gist-Brocades Nv Kluyveromyces as a host strain
US4560655A (en) 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4657866A (en) 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
US4620948A (en) 1982-12-22 1986-11-04 Genentech, Inc. Purification and activity assurance of precipitated heterologous proteins
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
AU3145184A (en) 1983-08-16 1985-02-21 Zymogenetics Inc. High expression of foreign genes in schizosaccharomyces pombe
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US5672347A (en) 1984-07-05 1997-09-30 Genentech, Inc. Tumor necrosis factor antagonists and their use
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US4879231A (en) 1984-10-30 1989-11-07 Phillips Petroleum Company Transformation of yeasts of the genus pichia
US4572798A (en) 1984-12-06 1986-02-25 Cetus Corporation Method for promoting disulfide bond formation in recombinant proteins
US4652630A (en) 1985-02-22 1987-03-24 Monsanto Company Method of somatotropin naturation
US5523215A (en) 1985-03-28 1996-06-04 Chiron Corporation Enhanced purification and expression of insoluble recombinant proteins
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
US4676980A (en) 1985-09-23 1987-06-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target specific cross-linked heteroantibodies
US5091178A (en) 1986-02-21 1992-02-25 Oncogen Tumor therapy with biologically active anti-tumor antibodies
US4927762A (en) 1986-04-01 1990-05-22 Cell Enterprises, Inc. Cell culture medium with antioxidant
GB8610600D0 (en) 1986-04-30 1986-06-04 Novo Industri As Transformation of trichoderma
IL85035A0 (en) 1987-01-08 1988-06-30 Int Genetic Eng Polynucleotide molecule,a chimeric antibody with specificity for human b cell surface antigen,a process for the preparation and methods utilizing the same
IL87737A (en) 1987-09-11 1993-08-18 Genentech Inc Method for culturing polypeptide factor dependent vertebrate recombinant cells
US5091313A (en) 1988-08-05 1992-02-25 Tanox Biosystems, Inc. Antigenic epitopes of IgE present on B cell but not basophil surface
US5720937A (en) 1988-01-12 1998-02-24 Genentech, Inc. In vivo tumor detection assay
US5506126A (en) 1988-02-25 1996-04-09 The General Hospital Corporation Rapid immunoselection cloning method
WO1989008114A1 (en) 1988-02-25 1989-09-08 The General Hospital Corporation Rapid immunoselection cloning method
US4861579A (en) 1988-03-17 1989-08-29 American Cyanamid Company Suppression of B-lymphocytes in mammals by administration of anti-B-lymphocyte antibodies
DE68919361T2 (de) 1988-06-21 1995-05-24 Genentech Inc Therapeutische zusammensetzungen für die behandlung von myocard-infarkten.
DE68925971T2 (de) 1988-09-23 1996-09-05 Cetus Oncology Corp Zellenzuchtmedium für erhöhtes zellenwachstum, zur erhöhung der langlebigkeit und expression der produkte
US6048728A (en) 1988-09-23 2000-04-11 Chiron Corporation Cell culture medium for enhanced cell growth, culture longevity, and product expression
US4981952A (en) 1988-10-04 1991-01-01 Eli Lilly And Company Method for the purification of vitamin K-dependent proteins
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
DE68927344T2 (de) 1989-04-28 1997-02-20 Rhein Biotech Proz & Prod Gmbh Hefezellen der Gattung-Schwanniomyces
EP0402226A1 (en) 1989-06-06 1990-12-12 Institut National De La Recherche Agronomique Transformation vectors for yeast yarrowia
DE3920358A1 (de) 1989-06-22 1991-01-17 Behringwerke Ag Bispezifische und oligospezifische, mono- und oligovalente antikoerperkonstrukte, ihre herstellung und verwendung
DE69029036T2 (de) 1989-06-29 1997-05-22 Medarex Inc Bispezifische reagenzien für die aids-therapie
FR2649120B1 (fr) 1989-06-30 1994-01-28 Cayla Nouvelle souche et ses mutants de champignons filamenteux, procede de production de proteines recombinantes a l'aide de ladite souche et souches et proteines obtenues selon ce procede
WO1991001367A1 (en) 1989-07-20 1991-02-07 Bioeng, Inc. Supercritical fluid disruption of and extraction from microbial cells
ATE119938T1 (de) 1989-08-21 1995-04-15 Takeda Chemical Industries Ltd Produktion von menschlichen nervenwachstumsfaktoren.
EP0676470A1 (en) 1989-10-16 1995-10-11 Amgen Inc. Stem cell factor
GB2237288A (en) 1989-10-18 1991-05-01 Celltech Ltd Amplification of a heterologous gene in recombinant eukaryotic host cells
DE69120146T2 (de) 1990-01-12 1996-12-12 Cell Genesys Inc Erzeugung xenogener antikörper
JPH06500011A (ja) 1990-06-29 1994-01-06 ラージ　スケール　バイオロジー　コーポレイション 形質転換された微生物によるメラニンの製造
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
ES2246502T3 (es) 1990-08-29 2006-02-16 Genpharm International, Inc. Animales no humanos transgenicos capaces de producir anticuerpos heterologos.
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
GB2251249B (en) 1990-12-28 1995-06-21 Mogam Biotech Res Inst High-density medium for animal cell culture
EP0586505A1 (en) 1991-05-14 1994-03-16 Repligen Corporation Heteroconjugate antibodies for treatment of hiv infection
ES2206447T3 (es) 1991-06-14 2004-05-16 Genentech, Inc. Anticuerpo humanizado para heregulina.
GB9114948D0 (en) 1991-07-11 1991-08-28 Pfizer Ltd Process for preparing sertraline intermediates
CA2113813C (en) 1991-08-14 2005-04-12 Paula M. Jardieu Immunoglobulin variants for specific fc epsilon receptors
WO1993008829A1 (en) 1991-11-04 1993-05-13 The Regents Of The University Of California Compositions that mediate killing of hiv-infected cells
WO1993010260A1 (en) 1991-11-21 1993-05-27 The Board Of Trustees Of The Leland Stanford Junior University Controlling degradation of glycoprotein oligosaccharides by extracellular glycosisases
AU675929B2 (en) 1992-02-06 1997-02-27 Curis, Inc. Biosynthetic binding protein for cancer marker
AU687346B2 (en) 1992-06-30 1998-02-26 Oncologix, Inc. A combination of anti-erbB-2 monoclonal antibodies and method of using
US5456909A (en) 1992-08-07 1995-10-10 T Cell Sciences, Inc. Glycoform fractions of recombinant soluble complement receptor 1 (sCR1) having extended half-lives in vivo
ATE149570T1 (de) * 1992-08-17 1997-03-15 Genentech Inc Bispezifische immunoadhesine
MX9305070A (es) 1992-08-21 1994-04-29 Genentech Inc Compocicion farmaceutica que contiene un antagonista de lfa-1 para el tratamiento de transtornos o desordenes mediados por el lfa-1
CA2149329C (en) 1992-11-13 2008-07-15 Darrell R. Anderson Therapeutic application of chimeric and radiolabeled antibodies to human b lymphocyte restricted differentiation antigen for treatment of b cell lymphoma
US7744877B2 (en) 1992-11-13 2010-06-29 Biogen Idec Inc. Expression and use of anti-CD20 Antibodies
US5736137A (en) 1992-11-13 1998-04-07 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5581476A (en) 1993-01-28 1996-12-03 Amgen Inc. Computer-based methods and articles of manufacture for preparing G-CSF analogs
US5417972A (en) 1993-08-02 1995-05-23 The Board Of Trustees Of The Leland Stanford Junior University Method of killing B-cells in a complement independent and an ADCC independent manner using antibodies which specifically bind CDIM
US5663304A (en) 1993-08-20 1997-09-02 Genentech, Inc. Refolding of misfolded insulin-like growth factor-I
US5595721A (en) 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
WO1995016203A2 (en) 1993-12-10 1995-06-15 Genentech, Inc. Methods for diagnosis of allergy and screening of anti-allergy therapeutics
DK0739214T3 (da) 1994-01-18 1998-10-07 Genentech Inc Fremgangsmåde til behandling af parasitinfektionerunder anvendelse af IgE antagonister
WO1995023865A1 (en) 1994-03-03 1995-09-08 Genentech, Inc. Anti-il-8 monoclonal antibodies for treatment of inflammatory disorders
GB9413127D0 (en) 1994-06-30 1994-08-24 Philips Electronics Uk Ltd Data processing apparatus
US5639635A (en) 1994-11-03 1997-06-17 Genentech, Inc. Process for bacterial production of polypeptides
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
IL117645A (en) 1995-03-30 2005-08-31 Genentech Inc Vascular endothelial cell growth factor antagonists for use as medicaments in the treatment of age-related macular degeneration
DE19513676A1 (de) * 1995-04-11 1996-10-17 Behringwerke Ag Cytoplasmatische Expression von Antikörpern, Antikörperfragmenten und Antikörperfragmentfusionsmolekülen in E.coli
ES2304786T3 (es) 1995-04-27 2008-10-16 Amgen Fremont Inc. Anticuerpos anti-il-8 humanos, derivados a partir de xenoratones inmunizados.
EP0823941A4 (en) 1995-04-28 2001-09-19 Abgenix Inc HUMAN ANTIBODIES DERIVED FROM IMMUNIZED XENO MOUSES
WO1996036358A1 (en) * 1995-05-15 1996-11-21 Cedars-Sinai Medical Center Compositions and methods for inhibiting xenograft rejection
US5721121A (en) 1995-06-06 1998-02-24 Genentech, Inc. Mammalian cell culture process for producing a tumor necrosis factor receptor immunoglobulin chimeric protein
US5830761A (en) 1995-06-07 1998-11-03 Genetics Institute, Inc. Medium and methods for culturing mammalian cho cells
CA2222231A1 (en) 1995-06-07 1996-12-19 Imclone Systems Incorporated Antibody and antibody fragments for inhibiting the growth of tumors
US5783186A (en) 1995-12-05 1998-07-21 Amgen Inc. Antibody-induced apoptosis
US5854067A (en) * 1996-01-19 1998-12-29 Board Of Regents, The University Of Texas System Hexokinase inhibitors
EP1297847A3 (en) 1996-01-23 2003-05-07 Genentech, Inc. Anti-CD18 antibodies in stroke
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
US5912327A (en) 1996-09-30 1999-06-15 Human Genome Sciences, Inc. Method of purifying chemokines from inclusion bodies
WO1998017321A1 (en) 1996-10-24 1998-04-30 Vanderbilt University Gene delivery and expression in areas inaccessible to direct protein delivery
AU729459B2 (en) 1996-11-15 2001-02-01 Genentech Inc. Purification of neurotrophins
IL130143A0 (en) 1996-11-27 2000-06-01 Genentech Inc Humanized anti-CD11a antibodies
EP1864999B1 (en) * 1996-11-27 2009-03-18 Genentech, Inc. Affinity purification of polypeptide on protein a matrix
US6037454A (en) 1996-11-27 2000-03-14 Genentech, Inc. Humanized anti-CD11a antibodies
DK1500329T3 (da) 1996-12-03 2012-07-09 Amgen Fremont Inc Humane antistoffer, der specifikt binder TNF-alfa
US6306393B1 (en) 1997-03-24 2001-10-23 Immunomedics, Inc. Immunotherapy of B-cell malignancies using anti-CD22 antibodies
US20020032315A1 (en) * 1997-08-06 2002-03-14 Manuel Baca Anti-vegf antibodies
EP0973804B1 (en) 1997-04-07 2006-12-27 Genentech, Inc. Anti-vegf antibodies
PT1860187E (pt) 1997-05-15 2011-10-04 Genentech Inc Receptor apo-2
EP0999853B1 (en) 1997-06-13 2003-01-02 Genentech, Inc. Stabilized antibody formulation
US6171586B1 (en) 1997-06-13 2001-01-09 Genentech, Inc. Antibody formulation
US6955917B2 (en) 1997-06-20 2005-10-18 Bayer Healthcare Llc Chromatographic method for high yield purification and viral inactivation of antibodies
DE69830315T2 (de) 1997-06-24 2006-02-02 Genentech Inc., San Francisco Galactosylierte glykoproteine enthaltende zusammensetzungen und verfahren zur deren herstellung
US6368596B1 (en) 1997-07-08 2002-04-09 Board Of Regents, The University Of Texas System Compositions and methods for homoconjugates of antibodies which induce growth arrest or apoptosis of tumor cells
ATE419009T1 (de) 1997-10-31 2009-01-15 Genentech Inc Methoden und zusammensetzungen bestehend aus glykoprotein-glykoformen
KR100253916B1 (ko) 1997-12-29 2000-05-01 김충환 사람 인슐린 전구체의 제조방법
BR9907990A (pt) 1998-01-30 2000-10-24 Genesense Technologies Inc Sequência de oligonucleotideos complementares aos genes tiorredoxina ou tiorredoxina reductase e métodos de utilização dos mesmos para modular o crescimento de células
ATE375365T1 (de) 1998-04-02 2007-10-15 Genentech Inc Antikörper varianten und fragmente davon
US6528624B1 (en) 1998-04-02 2003-03-04 Genentech, Inc. Polypeptide variants
US6242195B1 (en) 1998-04-02 2001-06-05 Genentech, Inc. Methods for determining binding of an analyte to a receptor
US6194551B1 (en) 1998-04-02 2001-02-27 Genentech, Inc. Polypeptide variants
AU3657899A (en) 1998-04-20 1999-11-08 James E. Bailey Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
PT1071752E (pt) 1998-04-21 2003-11-28 Micromet Ag Polipeptidos especificos para cd19xcd3 e suas utilizacoes
EP1241250A3 (en) 1998-05-15 2003-03-19 Genentech, Inc. Human dehydrogenase-like protein and nucleic acid encoding it
EP1112084B2 (en) 1998-08-11 2012-04-25 Biogen Idec Inc. Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody
AU739394B2 (en) 1998-08-27 2001-10-11 Lg Chemical Limited Process for the preparation of active somatotropin from inclusion bodies
MXPA01002674A (es) * 1998-09-14 2002-06-04 Ostergaard Pedersen Lars Un metodo para producir una proteina de la superfamilia de inmunoglobulina funcional.
US6224866B1 (en) 1998-10-07 2001-05-01 Biocrystal Ltd. Immunotherapy of B cell involvement in progression of solid, nonlymphoid tumors
MY155913A (en) 1998-11-09 2015-12-15 Biogen Inc Chimeric anti-cd20 antibody treatment of patients receiving bmt or pbsc transpants
WO2000027428A1 (en) 1998-11-09 2000-05-18 Idec Pharmaceuticals Corporation Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody
HUP0104865A3 (en) 1999-01-15 2004-07-28 Genentech Inc Polypeptide variants with altered effector function
US6897044B1 (en) 1999-01-28 2005-05-24 Biogen Idec, Inc. Production of tetravalent antibodies
US6383276B1 (en) 1999-03-12 2002-05-07 Fuji Photo Film Co., Ltd. Azomethine compound and oily magenta ink
EP1048732A1 (de) * 1999-04-26 2000-11-02 F. Hoffmann-La Roche Ag Verfahren zur Herstellung von natürlich gefalteten und sekretierten Proteinen
WO2000067796A1 (en) 1999-05-07 2000-11-16 Genentech, Inc. Treatment of autoimmune diseases with antagonists which bind to b cell surface markers
US7074403B1 (en) 1999-06-09 2006-07-11 Immunomedics, Inc. Immunotherapy of autoimmune disorders using antibodies which target B-cells
US6946129B1 (en) 1999-06-08 2005-09-20 Seattle Genetics, Inc. Recombinant anti-CD40 antibody and uses thereof
ITMI991299A1 (it) 1999-06-11 2000-12-11 Consiglio Nazionale Ricerche Uso di anticorpi contro antigeni di superficie per il trattamento della malattia trapianto contro ospite
DE19930748C2 (de) 1999-07-02 2001-05-17 Infineon Technologies Ag Verfahren zur Herstellung von EEPROM- und DRAM-Grabenspeicherzellbereichen auf einem Chip
JP2003528805A (ja) 1999-07-12 2003-09-30 ジェネンテック・インコーポレーテッド Ｃｄ２０に結合するアンタゴニストを用いた異種抗原に対する免疫応答のブロッキング
AU778022B2 (en) 1999-07-30 2004-11-11 Genentech Inc. Charged filtration membranes and uses therefor
US8557244B1 (en) 1999-08-11 2013-10-15 Biogen Idec Inc. Treatment of aggressive non-Hodgkins lymphoma with anti-CD20 antibody
US6451284B1 (en) 1999-08-11 2002-09-17 Idec Pharmaceuticals Corporation Clinical parameters for determining hematologic toxicity prior to radioimmunotheraphy
CN100389825C (zh) 1999-08-11 2008-05-28 拜奥根Idec公司 用抗cd20抗体治疗累及骨髓的非霍奇金淋巴瘤患者
WO2001013945A1 (en) 1999-08-23 2001-03-01 Biocrystal Ltd. Methods and compositions for immunotherapy of b cell involvement in promotion of a disease condition comprising multiple sclerosis
AUPQ296799A0 (en) 1999-09-20 1999-10-14 Unisearch Limited A cell-membrane impermeable trivalent organoarsenical derivative and use thereof
US6872563B1 (en) 1999-10-05 2005-03-29 President And Fellows Of Harvard College Compositions and methods for production of disulfide bond containing proteins in host cells
IL149116A0 (en) 1999-10-29 2002-11-10 Genentech Inc Anti-prostate stem cell antigen (psca) antibody compositions and methods of use
US20020006404A1 (en) 1999-11-08 2002-01-17 Idec Pharmaceuticals Corporation Treatment of cell malignancies using combination of B cell depleting antibody and immune modulating antibody related applications
US6808902B1 (en) 1999-11-12 2004-10-26 Amgen Inc. Process for correction of a disulfide misfold in IL-1Ra Fc fusion molecules
US6426208B1 (en) 1999-11-12 2002-07-30 Harbor-Ucla Research And Education Institute Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
JP2003530316A (ja) 1999-12-22 2003-10-14 ノボ ノルディスク アクティーゼルスカブ スクランブルした一本鎖ポリペプチドの抽出的再生法
CA2404390A1 (en) 2000-03-24 2001-10-04 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma using a combination of an antibody to cd20 and interleukin-2
US20030185796A1 (en) 2000-03-24 2003-10-02 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
KR20020091170A (ko) 2000-03-31 2002-12-05 아이덱 파마슈티칼즈 코포레이션 Ｂ 세포 림프종의 치료를 위한 항-사이토카인 항체 또는길항제 및 항-ｃｄ２０의 조합된 사용
ES2528794T3 (es) 2000-04-11 2015-02-12 Genentech, Inc. Anticuerpos multivalentes y usos de los mismos
CN1437478A (zh) 2000-04-25 2003-08-20 Idec药物公司 瑞图希单抗的鞘内施用,用于中枢神经系统淋巴瘤的治疗
US20030009301A1 (en) 2000-05-30 2003-01-09 M.B. Anand Integrated utility meter-reading, billing, payment and usage management system
WO2001097858A2 (en) 2000-06-20 2001-12-27 Idec Pharmaceuticals Corporation Cold anti-cd20 antibody/radiolabeled anti-cd22 antibody combination
WO2001097843A2 (en) 2000-06-22 2001-12-27 University Of Iowa Research Foundation Methods for enhancing antibody-induced cell lysis and treating cancer
JP2004502742A (ja) 2000-07-12 2004-01-29 アイデック　ファーマスーティカルズ　コーポレイション Ｂ細胞を消滅させる抗体及び免疫調節抗体を併用するｂ細胞悪性疾患の治療関連出願
US7041479B2 (en) * 2000-09-06 2006-05-09 The Board Of Trustess Of The Leland Stanford Junior University Enhanced in vitro synthesis of active proteins containing disulfide bonds
US6548276B2 (en) 2000-09-06 2003-04-15 The Board Of Trustees Of The Leland Stanford Junior University Enhanced in vitro synthesis of active proteins containing disulfide bonds
CA2422076A1 (en) 2000-09-18 2002-03-21 Idec Pharmaceutical Corporation Combination therapy for treatment of autoimmune diseases using b cell depleting/immunoregulatory antibody combination
US20040185535A1 (en) 2003-03-21 2004-09-23 Giles Wilson Industrial-scale serum-free production of recombinant FVII in mammalian cells
AU2002213357A1 (en) 2000-10-20 2002-05-06 Idec Pharmaceuticals Corporation Variant igg3 rituxan r and therapeutic use thereof
US20070292411A1 (en) 2000-11-08 2007-12-20 Human Genome Sciences, Inc. Antibodies That Immunospecifically Bind to TRAIL Receptors
WO2002096948A2 (en) 2001-01-29 2002-12-05 Idec Pharmaceuticals Corporation Engineered tetravalent antibodies and methods of use
WO2002060955A2 (en) 2001-01-29 2002-08-08 Idec Pharmaceuticals Corporation Modified antibodies and methods of use
US20030103971A1 (en) 2001-11-09 2003-06-05 Kandasamy Hariharan Immunoregulatory antibodies and uses thereof
CA2438094C (en) 2001-02-23 2011-10-11 Immunex Corporation Increased recovery of active proteins
US7332289B2 (en) 2001-03-09 2008-02-19 Chugai Seiyaku Kabushiki Kaisha Method of purifying protein
US20030040095A1 (en) 2001-03-16 2003-02-27 Achille Arini Method for the production of pharmaceutically active recombinant proteins
DE60239931D1 (de) 2001-04-02 2011-06-16 Genentech Inc Kombinationstherapie
US20030003097A1 (en) 2001-04-02 2003-01-02 Idec Pharmaceutical Corporation Recombinant antibodies coexpressed with GnTIII
WO2003061694A1 (en) 2001-05-10 2003-07-31 Seattle Genetics, Inc. Immunosuppression of the humoral immune response by anti-cd20 antibodies
AU2002316230A1 (en) 2001-06-13 2002-12-23 Genentech, Inc. Methods of culturing animal cells and polypeptide production in animal cells
JP2005515161A (ja) 2001-06-14 2005-05-26 インターミューン インコーポレイテッド γ−インターフェロンおよびＢ細胞特異的抗体の併用療法
US7321026B2 (en) 2001-06-27 2008-01-22 Skytech Technology Limited Framework-patched immunoglobulins
NZ592087A (en) 2001-08-03 2012-11-30 Roche Glycart Ag Antibody glycosylation variants having increased antibody-dependent cellular cytotoxicity
EP2131198B1 (en) 2001-09-20 2013-03-27 Board of Regents, The University of Texas System Measuring circulating therapeutic antibody, antigen and antigen/antibody complexes using ELISA assays
CA2460206C (en) 2001-10-02 2012-01-24 Novo Nordisk Health Care Ag Method for production of recombinant proteins in eukaryote cells
ES2326964T3 (es) 2001-10-25 2009-10-22 Genentech, Inc. Composiciones de glicoproteina.
US6852680B2 (en) 2001-10-26 2005-02-08 Ethyl Corporation Dithiocarbamates containing alkylthio and hydroxy substituents
US20060040348A1 (en) 2001-11-13 2006-02-23 Minmin Qin Methods for producing and purifying recombinant alpha-L-iduronidase
AU2002362098A1 (en) 2001-12-07 2003-06-23 Chiron Corporation Methods of therapy for non-hodgkin's lymphoma
GB0130955D0 (en) 2001-12-24 2002-02-13 Cancer Res Ventures Expression system
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
EP1572902B1 (en) 2002-02-01 2014-06-11 Life Technologies Corporation HIGH POTENCY siRNAS FOR REDUCING THE EXPRESSION OF TARGET GENES
CA2476166C (en) 2002-02-14 2011-11-15 Immunomedics, Inc. Anti-cd20 antibodies and fusion proteins thereof and methods of use
WO2003083056A2 (en) 2002-03-23 2003-10-09 Research Development Foundation Secretion of proteins with multiple disulfide bonds in bacteria and uses thereof
CA2481837A1 (en) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
CA2478925C (en) 2002-04-26 2016-06-07 Robert Lee Fahrner Non-affinity purification of proteins
US20030219818A1 (en) 2002-05-10 2003-11-27 Bohen Sean P. Methods and compositions for determining neoplastic disease responsiveness to antibody therapy
US20040029229A1 (en) 2002-05-20 2004-02-12 Reeves Philip J. High level protein expression system
AU2002345829A1 (en) 2002-06-24 2004-01-06 Dr. Reddy's Laboratories Ltd. Process for preparing g-csf
GB0216648D0 (en) 2002-07-18 2002-08-28 Lonza Biologics Plc Method of expressing recombinant protein in CHO cells
US7189811B2 (en) 2002-09-06 2007-03-13 National Institute Of Immunology Process for solubilization of recombinant proteins expressed as inclusion body
ES2558303T3 (es) * 2002-09-11 2016-02-03 Chugai Seiyaku Kabushiki Kaisha Método de purificación de proteínas
AU2003286493A1 (en) 2002-10-18 2004-05-04 Abgenix, Inc. System and method for cleaving antibodies
EP1581644B1 (en) 2003-01-09 2007-06-06 Genentech, Inc. Purification of polypeptides
EP1592703B1 (en) 2003-01-31 2008-06-04 N.V. Organon Method for protein isolation in anoxic conditions
US20060235201A1 (en) * 2003-02-06 2006-10-19 Roman Kischel Enduring T cell response
ES2786568T3 (es) 2003-07-28 2020-10-13 Genentech Inc Reducción de la lixiviación de la proteína A durante la cromatografía de afinidad con proteína A
WO2005035748A1 (en) 2003-10-10 2005-04-21 Novo Nordisk Health Care Ag Method for large-scale production of a polypeptide in eukaryote cells and a culture vessel suitable therefor
WO2005083058A1 (en) 2004-03-01 2005-09-09 Ares Trading S.A. Use of a serum-free cell culture medium for the production of il-18bp in mammalian cells
EP1791565B1 (en) 2004-09-23 2016-04-20 Genentech, Inc. Cysteine engineered antibodies and conjugates
US7435804B2 (en) 2004-10-19 2008-10-14 Phage Biotechnology, Inc. Method for obtaining single chain antibodies to human interferon α2b
WO2006060083A1 (en) 2004-10-22 2006-06-08 Amgen Inc. Methods for refolding polypeptides
KR101370253B1 (ko) 2004-10-22 2014-03-05 암젠 인크 재조합 항체의 재접힘 방법
GB0425534D0 (en) 2004-11-19 2004-12-22 Celltech R&D Ltd Process for obtaining antibodies
JP2006186521A (ja) 2004-12-27 2006-07-13 Leader Electronics Corp チューナのビットエラーレート（ｂｅｒ）測定の方法および装置
US8029783B2 (en) 2005-02-02 2011-10-04 Genentech, Inc. DR5 antibodies and articles of manufacture containing same
DOP2006000029A (es) 2005-02-07 2006-08-15 Genentech Inc Antibody variants and uses thereof. (variantes de un anticuerpo y usos de las mismas)
CA2629306A1 (en) 2005-11-23 2007-05-31 Genentech, Inc. Methods and compositions related to b cell assays
EP1996697A2 (en) 2006-03-07 2008-12-03 Novartis AG Preventing norvaline and norleucine misincorporation in recombinant proteins
AU2007294731B2 (en) * 2006-09-13 2014-04-17 Abbvie Inc. Cell culture improvements
TWI442965B (zh) * 2006-11-01 2014-07-01 Biogen Idec Inc 使用低ｐＨ及二價陽離子分離生物巨分子的方法
CN101801994A (zh) 2006-12-29 2010-08-11 格洛斯特制药公司 制备Romidepsin
US8467449B2 (en) * 2007-01-08 2013-06-18 Qualcomm Incorporated CAVLC enhancements for SVC CGS enhancement layer coding
JP5456658B2 (ja) 2007-03-30 2014-04-02 メディミューン，エルエルシー 抗体製剤
WO2008135498A2 (en) 2007-05-04 2008-11-13 Novo Nordisk A/S Prevention of protein degradation in mammalian cell cultures
SI3597659T1 (sl) 2007-07-09 2023-04-28 Genentech, Inc. Preprečevanje redukcije disulfidne vezi med rekombinantno proizvodnjo polipeptidov
WO2009006948A1 (en) 2007-07-12 2009-01-15 Michael Laurence Murphy A hydroalcoholic fat emulsion and emulsifier for use therein
KR20120031083A (ko) 2009-07-24 2012-03-29 에프. 호프만-라 로슈 아게 교반기 시스템
JP5731726B1 (ja) 2014-06-26 2015-06-10 楽天株式会社 情報処理装置、情報処理方法及び情報処理プログラム
AU2019375412A1 (en) 2018-11-05 2021-06-03 Avista Pharma Solutions, Inc. Chemical compounds
IT201900007998A1 (it) 2019-06-04 2020-12-04 Benelli Armi Spa Calcio per arma

Patent Citations (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US524474A (en) * 1894-08-14 Seed-planter
US670534A (en) * 1900-07-27 1901-03-26 Oliver L Carmical Seeding-machine and fertilizer-distributer.
US1369649A (en) * 1920-01-02 1921-02-22 George A Gieseler Pneumatic conveying mechanism
US2062295A (en) * 1932-11-22 1936-12-01 Niagara Sprayer And Chemical C Crop duster
US2171205A (en) * 1937-08-26 1939-08-29 Louis A Zinke Powder distributing attachment for tractors
US2193738A (en) * 1938-05-21 1940-03-12 Nat Conveyors Co Inc Pneumatic conveyer
US2559183A (en) * 1946-12-10 1951-07-03 Lawton L Barnett Transplanter drill set
US2671690A (en) * 1948-02-21 1954-03-09 Ehrenkrook Karl Von Forced air sprayer and duster
US2937049A (en) * 1949-10-18 1960-05-17 Osawa Hirosaburo Method of and an apparatus for carrying coals out of a vertical shaft with the aid of heavy liquid in the coal mine
US2814531A (en) * 1956-04-27 1957-11-26 Clough Equipment Co Balanced pneumatic conveying systems for pulverulent material
US3207560A (en) * 1962-12-26 1965-09-21 Monsanto Co Unloading device
US3197261A (en) * 1964-03-09 1965-07-27 Simpson Herbert Corp System for feeding and transporting materials
US3386474A (en) * 1965-05-26 1968-06-04 Chemagro Corp Diverter valve for finely divided solids
US3515315A (en) * 1966-01-24 1970-06-02 Kidd Designs Ltd Archie Combined seed and fertilizer drills for agricultural use
US3387895A (en) * 1966-12-29 1968-06-11 Combustion Eng Non-clogging splitter unit for dividing the flow of fluid-conveyed material
US3548765A (en) * 1967-12-27 1970-12-22 Noget Gougis Sa Agricultural distributing implement
US3543704A (en) * 1968-08-05 1970-12-01 Deere & Co Seed planter
US4082364A (en) * 1974-08-27 1978-04-04 Waeschle Maschinenfabrik Gmbh Method and apparatus for charging bulk material to a plurality of receiving stations
US4036408A (en) * 1975-12-24 1977-07-19 Acf Industries, Incorporated Hopper having means for directing blasts of gas to a discharge outlet or the hopper
US4280419A (en) * 1977-03-21 1981-07-28 International Harvester Company Pneumatic system for conveying granular material
US4200412A (en) * 1978-04-20 1980-04-29 Dynamic Air Incorporated Control for pneumatic conveying system
US4264243A (en) * 1979-01-04 1981-04-28 Dundee Cement Company Constant vacuum barge unloading system
US4244522A (en) * 1979-08-31 1981-01-13 Deere & Company Liquid storage and distribution apparatus for agricultural implements
US4379664A (en) * 1980-07-11 1983-04-12 Prasco Super Seeder Ltd. Seed cup assembly
US4413935A (en) * 1981-06-29 1983-11-08 Combustion Engineering, Inc. Flow splitter for dividing a stream of pulverulent material into multiple streams
US4506704A (en) * 1981-12-30 1985-03-26 Shell Oil Company Distributing valve
US4553882A (en) * 1982-10-28 1985-11-19 Trutzschler Gmbh & Co. Kg Method and apparatus for pneumatically conveying fiber material
US4562779A (en) * 1984-01-26 1986-01-07 Briggs Manufacturing, Inc. Foldable drill implement
US4674922A (en) * 1984-06-02 1987-06-23 Bernd Federhen Apparatus and process for controlling a charging operation for a feed of particles of bulk material
US4738770A (en) * 1985-11-25 1988-04-19 Cities Service Oil And Gas Corp. Apparatus and method of removal of catalyst blockage from reactor recycle pumps
US4779765A (en) * 1987-04-13 1988-10-25 Deere & Company Fertilizer divider for dual air system
US4793743A (en) * 1987-04-29 1988-12-27 Morris Rod-Weeder Company Ltd. Air seeder purging system
US4872785A (en) * 1988-01-22 1989-10-10 Schrage Timothy L Shut-off means for air-actuated planter
US4843983A (en) * 1988-02-02 1989-07-04 Deere & Company Pneumatic manifold quick coupling
US5156102A (en) * 1988-11-16 1992-10-20 Kongskilde Maskinfabrik A/S Apparatus for sowing granular material
US5033914A (en) * 1989-09-29 1991-07-23 Cyclonaire Corporation High efficiency feeder apparatus for pneumatic conveying lines
US5069583A (en) * 1990-10-26 1991-12-03 Caldwell Lawrence G Device for changing flow paths in a pneumatic conveying system
US5494381A (en) * 1991-04-11 1996-02-27 The Young Industries, Inc. Apparatus and method for pneumatically conveying bulk materials
US5161473A (en) * 1991-05-17 1992-11-10 Deere & Company Seed distribution system and method for a seeding implement
US5240355A (en) * 1992-05-22 1993-08-31 Nol-Tec Systems, Inc. Dense phase transporter pneumatic conveying system
US5379706A (en) * 1993-04-07 1995-01-10 Agco Corporation Seed distribution system for planters and drills
US5392722A (en) * 1993-04-20 1995-02-28 Deere & Company Air separation tube for a pneumatic delivery system
US5407305A (en) * 1993-04-29 1995-04-18 Semco, Inc. Continuous dense phase conveying method utilizing high pressure gas at predetermined gas pressures within a conveying pipe
US5575225A (en) * 1995-02-27 1996-11-19 Unverferth Manufacturing Company, Inc. Air-powered apparatus for filling grain drills and method of using same
US5813801A (en) * 1996-06-28 1998-09-29 Mac Equipment, Inc. Dense phase particulate conveying system and method with continuous air leakage management
US5749682A (en) * 1996-09-10 1998-05-12 Renfro Corporation Article collection and distribution system
US5927217A (en) * 1997-06-16 1999-07-27 Conserva Pak Seeding Systems Metering particulate material
US5878679A (en) * 1997-08-18 1999-03-09 Deere & Company Product disconnect for metering device
US6047652A (en) * 1998-05-12 2000-04-11 Case Corporation Seed planter distribution system
US6253693B1 (en) * 1998-11-05 2001-07-03 Flexi-Coil Ltd. Nurse adapter for air seeders

Cited By (104)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090010921A1 (en) * 2003-11-05 2009-01-08 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
US9296820B2 (en) 2003-11-05 2016-03-29 Roche Glycart Ag Polynucleotides encoding anti-CD20 antigen binding molecules with increased Fc receptor binding affinity and effector function
US8883980B2 (en) 2003-11-05 2014-11-11 Roche Glycart Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
US8906646B2 (en) 2006-09-13 2014-12-09 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
US9284371B2 (en) 2006-09-13 2016-03-15 Abbvie Inc. Methods of producing adalimumab
US10119118B2 (en) 2006-09-13 2018-11-06 Abbvie Inc. Modified serum-free cell culture medium
US9090867B2 (en) 2006-09-13 2015-07-28 Abbvie Inc. Fed-batch method of making anti-TNF-alpha antibody
US9234032B2 (en) 2006-09-13 2016-01-12 Abbvie Inc. Fed-batch methods for producing adalimumab
US9073988B2 (en) 2006-09-13 2015-07-07 Abbvie Inc. Fed batch method of making anti-TNF-alpha antibodies
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
US11639395B2 (en) 2007-07-09 2023-05-02 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US10759866B2 (en) 2007-07-09 2020-09-01 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US10808037B1 (en) 2007-07-09 2020-10-20 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US8574869B2 (en) 2007-07-09 2013-11-05 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US10906986B2 (en) 2007-07-09 2021-02-02 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US11078294B2 (en) 2007-07-09 2021-08-03 Genentech, Inc. Prevention of disulfide bond reduction during recombinant production of polypeptides
US11414498B2 (en) 2008-01-30 2022-08-16 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US11597776B2 (en) 2008-01-30 2023-03-07 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US20100135987A1 (en) * 2008-10-20 2010-06-03 Hickman Robert K Isolation and purification of antibodies using protein a affinity chromatography
US8895709B2 (en) 2008-10-20 2014-11-25 Abbvie Inc. Isolation and purification of antibodies using protein A affinity chromatography
US9018361B2 (en) 2008-10-20 2015-04-28 Abbvie Inc. Isolation and purification of antibodies using protein a affinity chromatography
US9109010B2 (en) 2008-10-20 2015-08-18 Abbvie Inc. Viral inactivation during purification of antibodies cross reference to related applications
US9464138B2 (en) * 2009-04-30 2016-10-11 Ablynx N.V. Method for the production of domain antibodies
US20120157664A1 (en) * 2009-04-30 2012-06-21 Ablynx N.V. Method for the production of domain antibodies
US20170081692A1 (en) * 2009-04-30 2017-03-23 Ablynx N.V. Method for the production of domain antibodies
US20100322931A1 (en) * 2009-06-17 2010-12-23 Harding Fiona A Anti-vegf antibodies and their uses
US9079953B2 (en) 2009-06-17 2015-07-14 Abbvie Biotherapeutics Inc. Anti-VEGF antibodies and their uses
US10280227B2 (en) 2009-09-11 2019-05-07 Genentech, Inc. Highly concentrated pharmaceutical formulations
US10377831B2 (en) 2009-09-11 2019-08-13 Genentech, Inc. Highly concentrated pharmaceutical formulations
US10752696B2 (en) 2009-09-11 2020-08-25 Genentech, Inc. Highly concentrated pharmaceutical formulations
US20110076273A1 (en) * 2009-09-11 2011-03-31 Genentech, Inc. Highly Concentrated Pharmaceutical Formulations
US20110177095A1 (en) * 2009-12-16 2011-07-21 Abbott Biotherapeutics Corporation Anti-her2 antibodies and their uses
US8937159B2 (en) 2009-12-16 2015-01-20 Abbvie Biotherapeutics Inc. Anti-HER2 antibodies and their uses
US9085622B2 (en) 2010-09-03 2015-07-21 Glaxosmithkline Intellectual Property Development Limited Antigen binding proteins
US10344083B2 (en) 2010-12-01 2019-07-09 Alderbio Holdings Llc Anti-NGF compositions and use thereof
US10457727B2 (en) 2010-12-01 2019-10-29 Alderbio Holdings Llc Methods of preventing inflammation and treating pain using anti-NGF compositions
US8911734B2 (en) 2010-12-01 2014-12-16 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75
US11214610B2 (en) 2010-12-01 2022-01-04 H. Lundbeck A/S High-purity production of multi-subunit proteins such as antibodies in transformed microbes such as Pichia pastoris
US9539324B2 (en) 2010-12-01 2017-01-10 Alderbio Holdings, Llc Methods of preventing inflammation and treating pain using anti-NGF compositions
US9718882B2 (en) 2010-12-01 2017-08-01 Alderbio Holdings Llc Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with P75
US9067988B2 (en) 2010-12-01 2015-06-30 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
US9783602B2 (en) 2010-12-01 2017-10-10 Alderbio Holdings Llc Anti-NGF compositions and use thereof
US9078878B2 (en) 2010-12-01 2015-07-14 Alderbio Holdings Llc Anti-NGF antibodies that selectively inhibit the association of NGF with TrkA, without affecting the association of NGF with p75
US9738713B2 (en) 2010-12-01 2017-08-22 Alderbio Holdings Llc Methods of preventing or treating pain using anti-NGF antibodies
US10227402B2 (en) 2010-12-01 2019-03-12 Alderbio Holdings Llc Anti-NGF antibodies and anti-NGF antibody fragments
US10221236B2 (en) 2010-12-01 2019-03-05 Alderbio Holdings Llc Anti-NGF antibodies that selectively inhibit the association of NGF with TRKA without affecting the association of NGF with P75
US9884909B2 (en) 2010-12-01 2018-02-06 Alderbio Holdings Llc Anti-NGF compositions and use thereof
US9783601B2 (en) 2010-12-01 2017-10-10 Alderbio Holdings Llc Methods of preventing inflammation and treating pain using anti-NGF compositions
US9255143B2 (en) 2011-04-27 2016-02-09 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9365645B1 (en) 2011-04-27 2016-06-14 Abbvie, Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9090688B2 (en) 2011-04-27 2015-07-28 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9505834B2 (en) 2011-04-27 2016-11-29 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US9062106B2 (en) 2011-04-27 2015-06-23 Abbvie Inc. Methods for controlling the galactosylation profile of recombinantly-expressed proteins
US20200199639A1 (en) * 2012-03-27 2020-06-25 Genentech, Inc. Harvest operations for recombinant proteins
US9765379B2 (en) 2012-03-27 2017-09-19 Genentech, Inc. Harvest operations for recombinant proteins
US20140080180A1 (en) * 2012-03-27 2014-03-20 Genentech, Inc. Harvest operations for recombinant proteins
US9505833B2 (en) 2012-04-20 2016-11-29 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9334319B2 (en) 2012-04-20 2016-05-10 Abbvie Inc. Low acidic species compositions
US9150645B2 (en) 2012-04-20 2015-10-06 Abbvie, Inc. Cell culture methods to reduce acidic species
US9957318B2 (en) 2012-04-20 2018-05-01 Abbvie Inc. Protein purification methods to reduce acidic species
US9193787B2 (en) 2012-04-20 2015-11-24 Abbvie Inc. Human antibodies that bind human TNF-alpha and methods of preparing the same
US9683033B2 (en) 2012-04-20 2017-06-20 Abbvie, Inc. Cell culture methods to reduce acidic species
US9346879B2 (en) 2012-04-20 2016-05-24 Abbvie Inc. Protein purification methods to reduce acidic species
US9708400B2 (en) 2012-04-20 2017-07-18 Abbvie, Inc. Methods to modulate lysine variant distribution
US9359434B2 (en) 2012-04-20 2016-06-07 Abbvie, Inc. Cell culture methods to reduce acidic species
US9181572B2 (en) 2012-04-20 2015-11-10 Abbvie, Inc. Methods to modulate lysine variant distribution
US9249182B2 (en) 2012-05-24 2016-02-02 Abbvie, Inc. Purification of antibodies using hydrophobic interaction chromatography
US9206390B2 (en) 2012-09-02 2015-12-08 Abbvie, Inc. Methods to control protein heterogeneity
US9234033B2 (en) 2012-09-02 2016-01-12 Abbvie, Inc. Methods to control protein heterogeneity
US9290568B2 (en) 2012-09-02 2016-03-22 Abbvie, Inc. Methods to control protein heterogeneity
US9512214B2 (en) 2012-09-02 2016-12-06 Abbvie, Inc. Methods to control protein heterogeneity
US9976163B2 (en) 2012-10-03 2018-05-22 Kyowa Hakko Kirin Co., Ltd Method for preventing reduction of polypeptide by adding amino acid to culture solution
US9815893B2 (en) 2012-11-30 2017-11-14 Abbvie Biotherapeutics Inc. Anti-VEGF antibodies and their uses
US9499614B2 (en) 2013-03-14 2016-11-22 Abbvie Inc. Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides
US9708399B2 (en) 2013-03-14 2017-07-18 Abbvie, Inc. Protein purification using displacement chromatography
US9067990B2 (en) 2013-03-14 2015-06-30 Abbvie, Inc. Protein purification using displacement chromatography
US8921526B2 (en) 2013-03-14 2014-12-30 Abbvie, Inc. Mutated anti-TNFα antibodies and methods of their use
US9598667B2 (en) 2013-10-04 2017-03-21 Abbvie Inc. Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins
US9085618B2 (en) 2013-10-18 2015-07-21 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9181337B2 (en) 2013-10-18 2015-11-10 Abbvie, Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9315574B2 (en) 2013-10-18 2016-04-19 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9200070B2 (en) 2013-10-18 2015-12-01 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US8946395B1 (en) 2013-10-18 2015-02-03 Abbvie Inc. Purification of proteins using hydrophobic interaction chromatography
US9522953B2 (en) 2013-10-18 2016-12-20 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9200069B2 (en) 2013-10-18 2015-12-01 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9499616B2 (en) 2013-10-18 2016-11-22 Abbvie Inc. Modulated lysine variant species compositions and methods for producing and using the same
US9688752B2 (en) 2013-10-18 2017-06-27 Abbvie Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9266949B2 (en) 2013-10-18 2016-02-23 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same
US9017687B1 (en) 2013-10-18 2015-04-28 Abbvie, Inc. Low acidic species compositions and methods for producing and using the same using displacement chromatography
US9550826B2 (en) 2013-11-15 2017-01-24 Abbvie Inc. Glycoengineered binding protein compositions
US11155610B2 (en) 2014-06-28 2021-10-26 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
US10653794B2 (en) * 2015-08-12 2020-05-19 Pfizer Inc. Capped and uncapped antibody cysteines, and their use in antibody-drug conjugation
WO2017027861A1 (en) 2015-08-13 2017-02-16 Amgen Inc. Charged depth filtration of antigen-binding proteins
EP4209499A1 (en) 2015-08-13 2023-07-12 Amgen Inc. Charged depth filtration of antigen-binding proteins
US11066465B2 (en) 2015-12-30 2021-07-20 Kodiak Sciences Inc. Antibodies and conjugates thereof
CN114703244A (zh) * 2016-05-10 2022-07-05 豪夫迈·罗氏有限公司 重组产生多肽期间减少三硫键的方法
US11760777B2 (en) 2017-04-26 2023-09-19 Bristol-Myers Squibb Company Methods of antibody production that minimize disulfide bond reduction
CN110831964A (zh) * 2017-05-09 2020-02-21 百时美施贵宝公司 在抗体制造期间控制粉红色形成的方法
US20190119374A1 (en) * 2017-05-17 2019-04-25 Immunogen, Inc. Compositions and methods for antibody production
US10913795B2 (en) * 2017-05-17 2021-02-09 Immunogen, Inc. Compositions and methods for antibody production
US11945862B2 (en) 2017-05-17 2024-04-02 Immunogen, Inc. Compositions and methods for antibody production
US11555052B2 (en) 2017-10-02 2023-01-17 Bayer Healthcare Llc Methods for preventing disulfide bond reduction in cell culture harvest with selenite
CN111164102A (zh) * 2017-10-02 2020-05-15 拜耳医药保健有限公司 用亚硒酸防止细胞培养收获物中的二硫键还原的方法
US11912784B2 (en) 2019-10-10 2024-02-27 Kodiak Sciences Inc. Methods of treating an eye disorder

Also Published As

Publication number Publication date
HRP20191905T1 (hr) 2020-01-24
EP2188302A2 (en) 2010-05-26
EP4245766A3 (en) 2024-01-17
IL272591A (en) 2020-03-31
KR102055873B1 (ko) 2019-12-13
US10906986B2 (en) 2021-02-02
DK3327026T3 (da) 2019-10-28
AU2013203411C9 (en) 2017-02-09
PL3327026T3 (pl) 2020-02-28
JP6030079B2 (ja) 2016-11-24
TW202124409A (zh) 2021-07-01
JP5687057B2 (ja) 2015-03-18
EP2586788A1 (en) 2013-05-01
WO2009009523A2 (en) 2009-01-15
HUE037162T2 (hu) 2018-08-28
AU2013203411B2 (en) 2016-09-08
SI3597659T1 (sl) 2023-04-28
EP3327026B1 (en) 2019-08-21
JP2018111698A (ja) 2018-07-19
SI3327026T1 (sl) 2019-11-29
EP2586788B1 (en) 2017-11-29
US10808037B1 (en) 2020-10-20
SG10201913363VA (en) 2020-03-30
PL2586788T3 (pl) 2018-05-30
US20130017598A1 (en) 2013-01-17
IL259860A (en) 2018-07-31
CN107090017A (zh) 2017-08-25
TWI574974B (zh) 2017-03-21
CA2691692A1 (en) 2009-01-15
AU2008275229B2 (en) 2014-11-06
EP4245766A9 (en) 2024-01-24
EP3327026A1 (en) 2018-05-30
US20140128575A1 (en) 2014-05-08
KR101851580B1 (ko) 2018-04-25
TWI666217B (zh) 2019-07-21
CN101932591B (zh) 2018-02-16
FI3597659T3 (fi) 2023-05-03
US20190135935A1 (en) 2019-05-09
EP2188302B1 (en) 2017-11-01
US20200347145A1 (en) 2020-11-05
US20210095040A1 (en) 2021-04-01
SI2188302T1 (en) 2018-07-31
IL202934B (en) 2019-05-30
DK3597659T3 (da) 2023-04-03
SG10201406411XA (en) 2014-11-27
KR20180042457A (ko) 2018-04-25
BRPI0812603A2 (pt) 2015-03-03
EP4335863A2 (en) 2024-03-13
PL2188302T3 (pl) 2018-04-30
EP4245766A2 (en) 2023-09-20
JP2017046706A (ja) 2017-03-09
JP6721621B2 (ja) 2020-07-15
TW201713679A (zh) 2017-04-16
KR102262298B1 (ko) 2021-06-07
CA3113365A1 (en) 2009-01-15
CN112480255A (zh) 2021-03-12
TWI702225B (zh) 2020-08-21
AU2013203411A1 (en) 2013-05-02
CN101932591A (zh) 2010-12-29
EP4219522A3 (en) 2023-09-20
JP2020105180A (ja) 2020-07-09
AU2008275229A1 (en) 2009-01-15
TR201802088T4 (tr) 2018-03-21
ES2655474T3 (es) 2018-02-20
ES2659829T3 (es) 2018-03-19
MX354993B (es) 2018-03-28
SG10202005450PA (en) 2020-07-29
IL237755A0 (en) 2015-05-31
PT3327026T (pt) 2019-10-30
MX2010000196A (es) 2010-04-21
CA2691692C (en) 2021-05-18
JP2010533192A (ja) 2010-10-21
US20230365704A1 (en) 2023-11-16
EP3597659A1 (en) 2020-01-22
IL237754A0 (en) 2015-05-31
PL3597659T3 (pl) 2023-07-10
PT3597659T (pt) 2023-04-04
DE17196350T1 (de) 2018-12-27
HUE061746T2 (hu) 2023-08-28
US20170313780A1 (en) 2017-11-02
AU2013203411B9 (en) 2017-02-09
HUE037633T2 (hu) 2018-09-28
ES2941738T3 (es) 2023-05-25
LT3597659T (lt) 2023-05-10
KR20150042305A (ko) 2015-04-20
US10759866B2 (en) 2020-09-01
EP4219522A2 (en) 2023-08-02
IL246683A0 (en) 2016-08-31
KR101643514B1 (ko) 2016-07-27
IL237755B (en) 2020-02-27
TW200914462A (en) 2009-04-01
HRP20230461T3 (hr) 2023-07-21
US20200317801A1 (en) 2020-10-08
EP3597659B1 (en) 2023-03-01
US20230383004A1 (en) 2023-11-30
KR20160105539A (ko) 2016-09-06
ES2751022T3 (es) 2020-03-30
HRP20180673T1 (hr) 2018-06-01
HUE046992T2 (hu) 2020-04-28
LT3327026T (lt) 2019-11-11
KR20210021144A (ko) 2021-02-24
JP2014129358A (ja) 2014-07-10
BRPI0812603B1 (pt) 2021-06-29
TW201942127A (zh) 2019-11-01
US11078294B2 (en) 2021-08-03
KR20190140090A (ko) 2019-12-18
SI2586788T1 (en) 2018-07-31
AU2013203411C1 (en) 2017-01-19
US20210188993A1 (en) 2021-06-24
WO2009009523A3 (en) 2009-05-14
US11639395B2 (en) 2023-05-02
KR20100061442A (ko) 2010-06-07
US8574869B2 (en) 2013-11-05

Similar Documents

Publication Publication Date Title
US11078294B2 (en) Prevention of disulfide bond reduction during recombinant production of polypeptides
US20240132612A1 (en) Prevention of disulfide bond reduction during recombinant production of polypeptides
AU2018201301B2 (en) Prevention of disulfide bond reduction during recombinant production of polypeptides

Legal Events

Date Code Title Description
AS Assignment

Owner name: GENENTECH, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KAO, YUNG-HSIANG;LAIRD, MICHAEL W.;SCHMIDT, MELODY TREXLER;AND OTHERS;REEL/FRAME:021771/0042;SIGNING DATES FROM 20081017 TO 20081027

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION