US20090148435A1 - Antibody purification by cation exchange chromatography - Google Patents

Antibody purification by cation exchange chromatography Download PDF

Info

Publication number
US20090148435A1
US20090148435A1 US12/260,623 US26062308A US2009148435A1 US 20090148435 A1 US20090148435 A1 US 20090148435A1 US 26062308 A US26062308 A US 26062308A US 2009148435 A1 US2009148435 A1 US 2009148435A1
Authority
US
United States
Prior art keywords
antibody
cation exchange
buffer
composition
exchange material
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
US12/260,623
Other languages
English (en)
Inventor
Benedicte Andree Lebreton
Deborah Ann O'Connor
Aurelia Safta
Mandakini Sharma
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=40262967&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20090148435(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Genentech Inc filed Critical Genentech Inc
Priority to US12/260,623 priority Critical patent/US20090148435A1/en
Assigned to GENENTECH, INC. reassignment GENENTECH, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SAFTA, AURELIA, LEBRETON, BENEDICTE ANDREE, O'CONNOR, DEBORAH ANN, SHARMA, MANDAKINI
Publication of US20090148435A1 publication Critical patent/US20090148435A1/en
Priority to US14/531,880 priority patent/US9896478B2/en
Priority to US15/850,885 priority patent/US20180118781A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K1/18—Ion-exchange chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
    • 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
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against 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
    • C07K16/28—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
    • 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

Definitions

  • This invention relates generally to protein purification.
  • the invention relates to a method for purifying antibody from a composition comprising the antibody and at least one contaminant using cation exchange chromatography, wherein a high pH wash step is used to remove contaminants prior to eluting the desired antibody using an elution buffer with increased conductivity.
  • proteins are produced by cell culture, using either eukaryotic or prokaryotic cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cells typically used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors, usually supplied from preparations of animal serum. Separation of the desired protein from the mixture of compounds fed to the cells and from the by-products of the cells themselves to a purity sufficient for use as a human therapeutic poses a daunting challenge.
  • Procedures for purification of proteins from cell debris initially depend on the site of expression of the protein. Some proteins can be cased to be secreted directly from the cell into the surrounding growth media; others are made intracellularly.
  • 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 in the course of the protein production run.
  • 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
  • U.S. Pat. No. 5,110,913 refers to purifying an antibody in an aqueous solution by binding the antibody to an ion exchange resin at a first pH of 4.6, washing at a second pH of 5.5, and eluting the antibody at pH 6.5, wherein the ionic strength of the solutions of these three steps remains constant.
  • Zhang et al. refer to Q membrane, anion exchange chromatography of a human antibody (Zhang et al. “Q Membrane Chromatography Application for Human Antibody Purification Process,” Poster presented at BioProduction , Oct. 26-27. Kunststoff, Germany, 2004).
  • Other publications concerning protein purification include: Barnthouse et al. J. Biotech. 66: 125-136 (1998); Blank et al.
  • the invention herein concerns an improved method for cation exchange chromatography of antibodies in which a high pH wash step is used to remove contaminants prior to eluting the desired antibody product.
  • the process results, amongst other things, in improved removal of Chinese Hamster Ovary Proteins (CHOP) contaminants.
  • CHOP Chinese Hamster Ovary Proteins
  • the invention provides a method for purifying an antibody from a composition comprising the antibody and at least one contaminant, which method comprises the sequential steps of:
  • the antibody binds human CD20, such as rituximab, or binds human vascular endothelial growth factor (VEGF), such as bevacizumab.
  • human CD20 such as rituximab
  • VEGF vascular endothelial growth factor
  • the invention concerns a method for purifying an antibody that binds human CD20 from a composition comprising the antibody and one or more contaminants selected from the group consisting of Chinese Hamster Ovary Proteins (CHOP), leached protein A, DNA, and aggregated CD20 antibody, which method comprises the sequential steps of:
  • the invention relates to a method for purifying an antibody that binds human vascular endothelial growth factor (VEGF) from a composition comprising the antibody and one or more contaminants selected from the group consisting of a cell culture media component, garamycin, Chinese Hamster Ovary Proteins (CHOP), DNA, viral contaminant, and aggregated VEGF antibody, which method comprises the sequential steps of:
  • the invention also concerns a composition
  • a composition comprising rituximab in a buffer comprising about 25 mM HEPES, at a pH of about 7.8.
  • the invention provides a composition comprising bevacizumab in a buffer comprising about 25 mM MOPS at a pH of about 7.0.
  • FIGS. 1A and 1B provide the amino acid sequences of the heavy chain (SEQ ID No. 1) and light chain (SEQ ID No. 2) of rituximab antibody.
  • Each of the framework regions (FR1-4) and each of the CDR regions (CDR1-3) in each variable region are identified, as are the human gamma 1 heavy chain constant sequence and human kappa light chain constant sequence.
  • the variable heavy (VH) region is in SEQ ID No. 3.
  • the variable light (VL) region is in SEQ ID No. 4.
  • the sequence identifiers for the CDRs are: CDR H1 (SEQ ID No. 5), CDR H2 (SEQ ID No. 6), CDR H3 (SEQ ID No. 7), CDR L1 (SEQ ID No. 8), CDR L2 (SEQ ID No. 9), and CDR L3 (SEQ ID No. 10).
  • FIGS. 2A and 2B provide the amino acid sequences of the heavy chain (SEQ ID No. 11) and light chain (SEQ ID No. 12) of bevacizumab antibody.
  • the end of each variable region is indicated with ⁇ .
  • the variable heavy (VH) region is in SEQ ID No. 13.
  • the variable light (VL) region is in SEQ ID No. 14.
  • Each of the three CDRs in each variable region is underlined.
  • the sequence identifiers for the CDRs are: CDR H1 (SEQ ID No. 15), CDR H2 (SEQ ID No. 16), CDR H3 (SEQ ID No. 17), CDR L1 (SEQ ID No. 18), CDR L2 (SEQ ID No. 19), and CDR L3 (SEQ ID No. 20).
  • FIG. 3 provides a side-by-side comparison of host cell proteins removal by the cation exchange chromatography process of the improved rituximab process compared to the original process. Superior CHOP removal was achieved with the new process.
  • the composition may be “partially purified” (i.e. having been subjected to one or more purification steps) or may be obtained directly from a host cell or organism producing the antibody (e.g. the composition may comprise harvested cell culture fluid).
  • polypeptide refers generally to peptides and proteins having more than about ten amino acids.
  • the polypeptide is a mammalian protein, examples of which include: renin; 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 -be
  • a preferred polypeptide is an intact antibody or an antibody fragment that binds to human CD20, for example, rituximab; or an intact antibody or an antibody fragment that binds to human vascular endothelial growth factor (VEGF), for example bevacizumab.
  • VEGF vascular endothelial growth factor
  • a “contaminant” is a material that is different from the desired antibody product.
  • the contaminant includes, without limitation: host cell materials, such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid; a variant, fragment, aggregate or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component (e.g. garamycin; GENTAMYCIN®) etc.
  • host cell materials such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid; a variant, fragment, aggregate or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component (e.g. garamycin; GENTAMYCIN®) etc.
  • cation exchange material refers to a solid phase that is negatively charged and has free cations for exchange with cations in an aqueous solution passed over or through the solid phase.
  • the charge may be provided by attaching one or more charged ligands to the solid phase, e.g. by covalent linking.
  • the charge may be an inherent property of the solid phase (e.g. as is the case for silica, which has an overall negative charge).
  • Commercially available cation exchange materials include carboxy-methyl-cellulose, BAKERBOND ABXTM, sulphopropyl (SP) immobilized on agarose (e.g.
  • SP-SEPHAROSE FAST FLOWTM, SP-SEPHAROSE FAST FLOW XLTM or SP-SEPHAROSE HIGH PERFORMANCETM from GE Healthcare
  • CAPTO STM GE Healthcare
  • FRACTOGEL-SO3TM, FRACTOGEL-SE HICAPTM e.g. S-SEPHAROSE FAST FLOWTM from GE Healthcare
  • SUPER SPTM Tosoh Biosciences
  • a preferred cation exchange material herein comprises cross-linked poly(styrene-divinylbenzene) flow-through particles (solid phase) coated with a polyhydroxylated polymer functionalized with sulfopropyl groups (for example, POROS 50 HS® chromatography resin).
  • solid phase is meant a non-aqueous matrix to which one or more charged ligands can adhere.
  • the solid phase may be a purification column (including, without limitation, expanded bed and packed bed columns), a discontinuous phase of discrete particles, a membrane, or filter etc.
  • materials for forming the solid phase include polysaccharides (such as agarose and cellulose) and other mechanically stable matrices such as silica (e.g. controlled pore glass), poly(styrene-divinylbenzene), polyacrylamide, ceramic particles and derivatives of any of the above.
  • load herein refers to the composition loaded onto the cation exchange material.
  • the cation exchange material is equilibrated with an equilibration buffer prior to loading the composition which is to be purified.
  • 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).
  • an “equilibration buffer” is a buffer that is used to equilibrate the cation exchange material, prior to loading the composition comprising the antibody of interest and one or more contaminants onto the cation exchange material.
  • the pH of the equilibration buffer herein is in the range from about 5.0 to about 6.0, preferably about 5.5.
  • the conductivity of the equilibration buffer herein is in the range from about 1 to about 8 mS/cm, preferably from about 4 to about 8 mS/cm, and most preferably from about 5 to about 8 mS/cm.
  • the equilibration buffer comprises a salt, such as NaCl, for example, in an amount from about 40 mM to about 80 mM, preferably about 60 mM NaCl.
  • wash buffer is used herein to refer to the buffer that is passed over the cation exchange material following loading of a composition and prior to elution of the protein of interest.
  • the wash buffer may serve to remove one or more contaminants from the cation exchange material, without substantial elution of the desired antibody product.
  • a “first wash buffer” and a “second wash buffer” are used.
  • first wash buffer refers to a wash buffer having a pH increased relative to the pH of the load and/or equilibration buffer.
  • the first wash buffer may be used herein to elute one or more contaminants from the cation exchange material, without substantially eluting the antibody product of interest therefrom.
  • first should not be interpreted as excluding the use of one or more additional wash or other buffers between the load and the first wash buffer.
  • the pH of the first wash buffer herein is in the range from about 6.8 to about 9.0, preferably from about 7.0 to about 8.0, and most preferably pH about 7.0 or pH about 7.8.
  • the conductivity of the first wash buffer herein is in the range from about 0.01 to about 5 mS/cm, preferably from about 0.1 to about 3 mS/cm, and most preferably from about 0.2 to about 2 mS/cm.
  • the first wash buffer is substantially free of a salt (such as NaCl) therein.
  • second wash buffer for the purposes of this application refers to a wash buffer used after the first wash buffer to prepare the cation exchange material for elution of the antibody of interest.
  • the term “second” should not be interpreted as excluding the use of one or more additional wash or other buffers between the first wash buffer and second wash buffer.
  • the pH of the second wash buffer herein is in the range from about 5.0 to about 6.0, preferably about 5.5, and most preferably pH 5.5.
  • the conductivity of the second wash buffer herein is in the range from about 0.01 to about 5 mS/cm, preferably about 0.1 to about 3 mS/cm, and most preferably from about 0.5 to about 3.0 mS/cm.
  • Elution buffer is used to elute the antibody of interest from the solid phase.
  • the elution buffer has a substantially increased conductivity relative to that of the second wash buffer, such that the desired antibody product is eluted from the cation exchange material.
  • the conductivity of the elution buffer is substantially greater than that of the load and of each of the preceding buffers, namely of the equilibration buffer, first wash buffer, and second wash buffer.
  • substantially greater conductivity is meant, for example, that the buffer has a conductivity which is at least 2, 3, 4, 5 or 6 conductivity units (mS/cm) greater than that of the composition or buffer to which it is being compared.
  • the pH of the elution buffer is substantially the same as that of the equilibration and/or second wash buffer.
  • the pH of the elution buffer herein is in the range from about 5.0 to about 6.0, preferably about 5.5, and most preferably pH 5.5.
  • the conductivity of the elution buffer herein is in the range from about 10 mS/cm to about 100 mS/cm, preferably from about 12 mS/cm to about 30 mS/cm, and most preferably from about 12 to about 20 mS/cm.
  • Increased conductivity may be achieved by the addition of a salt, such as sodium chloride, sodium acetate, potassium chloride to the elution buffer.
  • the elution buffer comprises from about 100 to about 300 mM NaCl, preferably from about 150 mM to about 200 mM NaCl, for example about 175 mM NaCl or about 160 mM NaCl.
  • a “regeneration buffer” may be used to regenerate the cation exchange material such that it can be re-used.
  • the regeneration buffer has a conductivity and/or pH as required to remove substantially all contaminants and the antibody of interest from the cation exchange material.
  • conductivity refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Therefore, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity.
  • the basic unit of measure for conductivity is the Siemen (or mho), mho (mS/cm), and can be measured using a conductivity meter, such as various models of Orion conductivity meters.
  • electrolytic conductivity is the capacity of ions in a solution to carry electrical current
  • the conductivity of a solution may be altered by changing the concentration of ions therein.
  • concentration of a buffering agent and/or the concentration of a salt e.g. sodium chloride, sodium acetate, or potassium chloride
  • the salt concentration of the various buffers is modified to achieve the desired conductivity.
  • purifying an antibody from a composition comprising the antibody and one or more contaminants is meant increasing the degree of purity of the antibody in the composition by removing (completely or partially) at least one contaminant from the composition.
  • a “purification step” may be part of an overall purification process resulting in a “homogeneous” composition.
  • “Homogeneous” is used herein to refer to a composition comprising at least about 70% by weight of the antibody of interest, based on total weight of the composition, preferably at least about 80% by weight, more preferably at least about 90% by weight, even more preferably at least about 95% by weight.
  • binding a molecule to a cation exchange material is meant exposing the molecule to the cation exchange material under appropriate conditions (pH and/or conductivity) such that the molecule is reversibly immobilized in or on the cation exchange material by virtue of ionic interactions between the molecule and a charged group or charged groups of the cation exchange material.
  • washing is meant passing an appropriate buffer through or over the cation exchange material.
  • eluting a molecule (e.g. antibody or contaminant) from a cation exchange material is meant to remove the molecule therefrom.
  • the antibody to be purified herein is a recombinant antibody.
  • a “recombinant antibody” is one which has been produced in a host cell which has been transformed or transfected with nucleic acid encoding the antibody, or produces the antibody as a result of homologous recombination.
  • Transformation and “transfection” are used interchangeably to refer to the process of introducing nucleic acid into a cell. Following transformation or transfection, the nucleic acid may integrate into the host cell genome, or may exist as an extrachromosomal element.
  • the “host cell” includes a cell in in vitro cell culture as well as a cell within a host animal. Methods for recombinant production of polypeptides are described in U.S. Pat. No. 5,534,615, expressly incorporated herein by reference, for example.
  • a “variant” or “amino acid sequence variant” of a starting polypeptide is a polypeptide that comprises an amino acid sequence different from that of the starting polypeptide.
  • a variant will possess at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 98% sequence identity with the native polypeptide. Percentage sequence identity is determined, for example, by the Fitch et al., Proc. Natl. Acad. Sci. USA 80:1382-1386 (1983), version of the algorithm described by Needleman et al., J. Mol. Biol. 48:443-453 (1970), after aligning the sequences to provide for maximum homology.
  • Amino acid sequence variants of a polypeptide may be prepared by introducing appropriate nucleotide changes into DNA encoding the polypeptide, or by peptide synthesis. Such variants include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequence of the polypeptide of interest. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics.
  • the amino acid changes also may alter post-translational processing of the polypeptide, such as by changing the number or position of glycosylation sites.
  • antibody is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired binding specificity.
  • 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 non-polypeptide 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 polypeptides discussed above.
  • CD polypeptides such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family such as the EGF receptor (HER1), HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and av/b3 integrin including either a or b subunits thereof (e.g. anti-CD 11a, 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; polypeptide C etc.
  • HER1 EGF receptor
  • HER2 HER2
  • HER3 or HER4 receptor cell adhesion molecules
  • cell adhesion molecules such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM and av/b3 integrin including either a or b sub
  • 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.
  • antibodies to be purified herein include, but are not limited to: HER2 antibodies including trastuzumab (HERCEPTIN®) (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat. No. 5,725,856) and pertuzumab (OMNITARGTM) (WO01/00245); CD20 antibodies (see below); IL-8 antibodies (St John et al., Chest, 103:932 (1993), and International Publication No.
  • HERCEPTIN® trastuzumab
  • OMNITARGTM pertuzumab
  • CD20 antibodies see below
  • IL-8 antibodies St John et al., Chest, 103:932 (1993), and International Publication No.
  • VEGF or VEGF receptor antibodies including humanized and/or affinity matured VEGF antibodies such as the humanized VEGF antibody huA4.6.1 bevacizumab (AVASTIN®) and ranibizumab (LUCENTIS®) (Kim et al., Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331, published Oct. 15, 1998); PSCA antibodies (WO01/40309); CD11a antibodies including efalizumab (RAPTIVA®) (U.S. Pat. No. 5,622,700, WO 98/23761, Steppe et al., Transplant Intl.
  • RPTIVA® efalizumab
  • CD25 or Tac antibodies such as CHI-621 (SIMULECT®) and ZENAPAX® (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); CD4 antibodies such as the cM-7412 antibody (Choy et al. Arthritis Rheum 39(1):52-56 (1996)); CD52 antibodies such as CAMPATH-1H (ILEX/Berlex) (Riechmann et al. Nature 332:323-337 (1988)); Fc receptor antibodies such as the M22 antibody directed against Fc ⁇ RI as in Graziano et al. J. Immunol.
  • carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al. Cancer Res. 55(23Suppl): 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.
  • CD38 antibodies e.g. AT 13/5 (Ellis et al. J. Immunol. 155(2):925-937 (1995)); CD33 antibodies such as Hu M195 (Jurcic et al. Cancer Res 55(23 Suppl):5908s-5910s (1995)) and CMA-676 or CDP771; EpCAM antibodies such as 17-1A (PANOREX®); GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO®); RSV antibodies such as MEDI-493 (SYNAGIS®); CMV antibodies such as PROTOVIR®; HIV antibodies such as PRO542; hepatitis antibodies such as the Hep B antibody OSTAVIR®; CA 125 antibody OvaRex; idiotypic GD3 epitope antibody BEC2; ⁇ v ⁇ 3 antibody (e.g.
  • human renal cell carcinoman antibody such as ch-G250; ING-1; anti-human 17-1An antibody (3622W94); anti-human colorectal tumor antibody (A33); anti-human melanoman antibody R24 directed against GD3 ganglioside; anti-human squamous-cell carcinoma (SF-25); human leukocyte antigen (HLA) antibody such as Smart ID10 and the anti-HLA DR antibody Oncolym (Lym-1); CD37 antibody such as TRU 016 (Trubion); IL-21 antibody (Zymogenetics/Novo Nordisk); anti-B cell antibody (Impheron); B cell targeting MAb (Immunogen/Aventis); 1D09C3 (Morphosys/GPC); LymphoRad 131 (HGS); Lym-1 antibody, such as Lym-1Y-90 (USC) or anti-Lym-1 Oncolym (USC/Peregrine); LIF 226 (Enhanced Life
  • anti-complement antibody such as C5 antibody (e.g. eculizumab, 5G1.1; Alexion); oral formulation of human immunoglobulin (e.g. IgPO; Protein Therapeutics); IL-12 antibody such as ABT-874 (CAT/Abbott); Teneliximab (BMS-224818; BMS); CD40 antibodies, including S2C6 and humanized variants thereof (WO00/75348) and TNX 100 (Chiron/Tanox); TNF- ⁇ antibodies including cA2 or infliximab (REMICADEL®), CDP571, MAK-195, adalimumab (HUMIRATM), pegylated TNF- ⁇ antibody fragment such as CDP-870 (Celltech), D2E7 (Knoll), anti-TNF- ⁇ polyclonal antibody (e.g.
  • C5 antibody e.g. eculizumab, 5G1.1; Alexion
  • oral formulation of human immunoglobulin e.g
  • CD22 antibodies such as LL2 or epratuzumab (LYMPHOCIDE®; Immunomedics), including epratuzumab Y-90 and epratzumab I-131, Abiogen's CD22 antibody (Abiogen, Italy), CMC 544 (Wyeth/Celltech), combotox (UT Soutwestern), BL22 (NIH), and LympoScan Tc99 (Immunomedics).
  • the antibody that is purified herein is a naked, intact antibody which binds to human CD20, or a naked, intact antibody which binds to human VEGF.
  • CD20 antigen is an about 35-kDa, non-glycosylated phosphoprotein found on the surface of greater than 90% of B cells from peripheral blood or lymphoid organs. CD20 is present on both normal B cells as well as malignant B cells, but is not expressed on stem cells. Other names for CD20 in the literature include “B-lymphocyte-restricted antigen” and “Bp35”. The CD20 antigen is described in Clark et al., Proc. Natl. Acad. Sci . (USA) 82:1766 (1985), for example.
  • a “CD20 antibody antagonist” herein is an antibody that, upon binding to CD20 on B cells, destroys or depletes B cells in a subject and/or interferes with one or more B-cell functions, e.g., by reducing or preventing a humoral response elicited by the B cell.
  • the antibody antagonist preferably is able to deplete B cells (i.e., reduce circulating B-cell levels) in a subject treated therewith. Such depletion may be achieved via various mechanisms such antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), inhibition of B-cell proliferation and/or induction of B-cell death (e.g., via apoptosis).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • B cell depletion refers to a reduction in B cell levels in an animal or human generally after drug or antibody treatment, as compared to the level before treatment. B cell depletion can be partial or complete. B cell levels are measurable using well known techniques such as those described in Reff et al., Blood 83: 435-445 (1994), or U.S. Pat. No. 5,736,137 (Anderson et al.).
  • a mammal e.g. a normal primate
  • peripheral B-cell concentrations may be determined, e.g. by a FACS method that counts B cells.
  • CD20 antibodies examples include: “C2B8,” which is now called “rituximab” (“RITUXAN®”) (U.S. Pat. No. 5,736,137); the yttrium-[90]-labelled 2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” (ZEVALIN®) commercially available from IDEC Pharmaceuticals, Inc. (U.S. Pat. No. 5,736,137; 2B8 deposited with ATCC under accession no. HB11388 on Jun.
  • the preferred CD20 antibodies herein are chimeric, humanized, or human CD20 antibodies, more preferably rituximab, humanized 2H7, 2F2 (Hu-Max-CD20) human CD20 antibody (Genmab), and humanized A20 or IMMUN-106 antibody (Immunomedics).
  • the terms “rituximab,” “RITUXAN®,” and “C2B8” herein refer to a recombinant chimeric antibody which binds to the human CD20 antigen as described in U.S. Pat. No. 5,736,137, Anderson et al.
  • Such antibody preferably comprises a heavy chain comprising CDR H1 (SEQ ID No. 5), CDR H2 (SEQ ID No. 6), CDR H3 (SEQ ID No. 7), and a light chain, wherein the light chain preferably comprises CDR L1 (SEQ ID No. 8), CDR L2 (SEQ ID No. 9), and CDR L3 (SEQ ID No.
  • the heavy chain comprises a variable heavy (VH) region comprising SEQ ID No. 3 and a variable light (VL) region comprising SEQ ID No. 4; and most preferably comprises a heavy chain comprising SEQ ID No. 1 (with or without a C-terminal lysine residue), and a light chain, wherein the light chain preferably comprises SEQ ID No. 2.
  • VH variable heavy
  • VL variable light
  • the terms expressly include variant forms such as described in Moorhouse et al. J. Pharm Biomed. Anal. 16:593-603 (1997).
  • human VEGF refers to the 165-amino acid human vascular endothelial cell growth factor, and related 121-, 189-, and 206-amino acid vascular endothelial cell growth factors, as described by Leung et al., Science 246:1306 (1989), and Houck et al., Mol. Endocrin. 5:1806 (1991) together with the naturally occurring allelic and processed forms of those growth factors.
  • the present invention provides anti-VEGF antagonistic antibodies which are capable of inhibiting one or more of the biological activities of VEGF, for example, its mitogenic or angiogenic activity.
  • Antagonists of VEGF act by interfering with the binding of VEGF to a cellular receptor, by incapacitating or killing cells which have been activated by VEGF, or by interfering with vascular endothelial cell activation after VEGF binding to a cellular receptor. All such points of intervention by a VEGF antagonist shall be considered equivalent for purposes of this invention.
  • bevacizumab refers to a recombinant humanized monoclonal antibody which binds human vascular endothelial growth factor (VEGF) antigen (rhuMAb VEGF) as described in U.S. Pat. No. 7,169,901, Presta et al.
  • VEGF vascular endothelial growth factor
  • rhuMAb VEGF vascular endothelial growth factor antigen
  • Such antibody preferably comprises a heavy chain comprising CDR H1 (SEQ ID No. 15), CDR H2 (SEQ ID No. 16), CDR H3 (SEQ ID No. 17), and a light chain, wherein the light chain preferably comprises CDR L1 (SEQ ID No.
  • the heavy chain comprises a variable heavy (VH) region comprising SEQ ID No. 13 and a variable light (VL) region comprising SEQ ID No. 14; and preferably comprises a heavy chain comprising SEQ ID No. 11 (with or without a C-terminal lysine residue), and a light chain, wherein the light chain preferably comprises SEQ ID No. 12.
  • VH variable heavy
  • VL variable light
  • the terms expressly include variant forms that form during production of the recombinant antibody product.
  • the term “monoclonal antibody” as used herein 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 naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • 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 the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).
  • “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.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • J H antibody heavy-chain joining region
  • the monoclonal antibodies herein specifically include “chimeric” antibodies (immunoglobulins) 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)).
  • chimeric antibodies immunoglobulins 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
  • hypervariable region when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding.
  • the hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Polypeptides of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop” (i.e.
  • “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (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 capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the 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 of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • variable domains both light and heavy
  • FR human framework
  • 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.
  • Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Various techniques have been developed for the production of 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.
  • the antibody fragments can be isolated from the antibody phage libraries discussed above.
  • 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)).
  • the F(ab′) 2 is formed using the leucine zipper GCN4 to promote assembly of the F(ab′) 2 molecule.
  • 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.
  • Single-chain Fv” or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains which enables the sFv to form the desired structure for antigen binding.
  • sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
  • 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 ).
  • V H heavy chain variable domain
  • V L light chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
  • linear antibodies when used throughout this application refers to the antibodies described in Zapata et al. Polypeptide 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.
  • Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are usually from 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.
  • BsAbs include those with one arm directed against a tumor cell antigen and the other arm directed against a cytotoxic trigger molecule such as anti-Fc ⁇ RI/anti-CD15, anti-p185 HER2 /Fc ⁇ RIII (CD16), anti-CD3/anti-malignant B-cell (1D10), anti-CD3/anti-p185 HER2 , anti-CD3/anti-p97, anti-CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma), anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGF receptor/anti-CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM)/anti-CD3, anti-folate binding protein (FBP)/anti-CD3, anti-pan carcinoma associated antigen (AMOC-31)/anti-CD3; BsAbs with one arm which binds specifically
  • BsAbs for use in therapy of infectious diseases such as anti-CD3/anti-herpes simplex virus (HSV), anti-T-cell receptor:CD3 complex/anti-influenza, anti-Fc ⁇ R/anti-HIV; BsAbs for tumor detection in vitro or in vivo such as anti-CEA/anti-EOTUBE, anti-CEA/anti-DPTA, anti-p185 HER2 /anti-hapten; BsAbs as vaccine adjuvants; and BsAbs as diagnostic tools such as anti-rabbit IgG/anti-ferritin, anti-horse radish peroxidase (HRP)/anti-hormone, anti-somatostatin/anti-substance P, anti-HRP/anti-FITC, anti-CEA/anti- ⁇ -galactosidase.
  • HRP anti-horse radish peroxidase
  • HRP anti-somatostatin/anti-substance P
  • trispecific antibodies examples include anti-CD3/anti-CD4/anti-CD37, anti-CD3/anti-CD5/anti-CD37 and anti-CD3/anti-CD8/anti-CD37.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab′) 2 bispecific antibodies).
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).
  • naked antibody for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • an “intact antibody” herein is one which comprises two antigen binding regions, and an Fc region.
  • the intact antibody has a functional Fc region.
  • 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.
  • a “disorder” is any condition that would benefit from treatment with the antibody purified as described herein. This includes both chronic and acute disorders and diseases and those pathological conditions which predispose the mammal to the disorder in question.
  • label when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody.
  • the label may be itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • cytotoxic agent refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small-molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.
  • radioactive isotopes e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188
  • the invention herein provides methods for purifying an antibody from a composition (e.g. an aqueous solution) comprising the antibody and one or more contaminants.
  • the composition is generally one resulting from the recombinant production of the antibody, but may be that resulting from production of the antibody by peptide synthesis (or other synthetic means) or the antibody may be purified from a native source of the antibody.
  • the antibody binds human CD20 antigen, such as rituximab, or binds human VEGF antigen, such as bevacizumab.
  • the nucleic acid encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression.
  • DNA encoding the antibody 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 antibody).
  • Many vectors are available.
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence (e.g. as described in U.S. Pat. No. 5,534,615, specifically incorporated herein by reference).
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryotic cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia , e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella , e.g., Salmonella typhimurium, Serratia , e.g., Serratia marcescans , and Shigella , as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B.
  • E. coli 294 ATCC 31,446
  • E. coli B E. coli X1776
  • E. coli W3110 ATCC 27,325
  • eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
  • waltii ATCC 56,500
  • K. drosophilarum ATCC 36,906
  • K. thermotolerans K. marxianus
  • yarrowia EP 402,226
  • Pichia pastoris EP 183,070
  • Candida Trichoderma reesia
  • Neurospora crassa Schwanniomyces such as Schwanniomyces occidentalis
  • filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium , and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • a variety of viral strains for transfection are publicly available, e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.
  • mammalian host cell lines include, but are not limited to, monkey kidney CV1 cells transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cells (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); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
  • COS-7 monkey kidney CV1 cells transformed by SV40
  • human embryonic kidney cells (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
  • Chinese hamster ovary cells/-DHFR CHO, Urlaub et al., Proc.
  • 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); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2).
  • CHO cells are preferred for the expression of antibodies, and may be advantageously used to produce the antibodies purified in accordance with the present invention.
  • Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.
  • the host cells used to produce the antibody of this invention may 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), nucleotides (such as adenosine and thymidine), antibiotics (such as garamycin; GENTAMYCIN®), 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.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed cells (e.g. resulting from homogenization), is removed, for example, by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
  • the composition to be subjected to the purification method herein is a recombinantly produced antibody, preferably an intact antibody, expressed by a Chinese Hamster Ovary (CHO) recombinant host cell culture.
  • the composition has been subjected to at least one purification step prior to cation exchange chromatography.
  • the composition contains the antibody of interest and one or more contaminants, such as Chinese Hamster Ovary Proteins (CHOP); leached protein A; nucleic acid; a variant, fragment, aggregate or derivative of the desired antibody; another polypeptide; endotoxin; viral contaminant; cell culture media component (e.g. garamycin; GENTAMYCIN®), etc.
  • Examples of additional purification procedures which may be performed prior to, during, or following the cation exchange chromatography method include fractionation on a hydrophobic interaction chromatography (e.g. on PHENYL-SEPHAROSETM), ethanol precipitation, isoelectric focusing, Reverse Phase HPLC, chromatography on silica, chromatography on HEPARIN SEPHAROSETM, anion exchange chromatography, further cation exchange chromatography, mixed-mode ion exchange, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, hydrophic charge induction chromatography, and affinity chromatography (e.g. using protein A, protein G, an antibody, or a specific substrate, ligand or antigen as the capture reagent).
  • affinity chromatography e.g. using protein A, protein G, an antibody, or a specific substrate, ligand or antigen as the capture reagent.
  • the cation exchange purification scheme typically includes the following steps performed sequentially: (1) equilibration of the cation exchange material; (2) loading the composition to be purified onto the cation exchange material, (3) a first wash step; (4) a second wash step, and (5) elution of the antibody of interest.
  • the efficacy of purification can be significantly improved.
  • performing the first wash step using a wash buffer with a pH in the range from about 6.8 to about 9.0 (e.g. from about 7.0 to 8.0), such as, for example, about pH 7.8 or about pH 7.0, contaminants as described above are removed more efficiently than using the conventional lower pH range of about 5.0 to about 5.5.
  • the host cell protein content of the composition comprising the antibody eluted from the cation exchange material is typically less than about 200 ppm, which is below the approximately 500 ppm level achieved using one wash step at a pH of about 5 to 5.5.
  • the cation exchange material comprises cross-linked poly(styrene-divinylbenzene) flow-through particles (solid phase) coated with a polyhydroxylated polymer functionalized with sulfopropyl groups, for example, a POROS 50 HS® column available from Applied Biosystems.
  • an equilibration buffer is passed over or through the cation exchange material prior to loading the composition comprising the antibody of interest and one or more contaminants onto the material.
  • the equilibration buffer has a pH from about 5.0 to about 6.0, for example about pH 5.5.
  • One exemplary equilibration buffer comprises 19 mM MES, 60 mM NaCl, pH 5.50.
  • Another exemplary equilibration buffer comprises 23 mM MES, 60 mM NaCl, pH 5.50.
  • an aqueous solution comprising the antibody of interest and one or more contaminants is loaded onto the cation exchange material.
  • the pH of the load is in the range from about 4.0 to about 6.0, for example about pH 5.0 or about pH 5.5.
  • a conditioned product pool from a prior purification step is loaded.
  • a Protein A pool from a prior Protein A chromatography purification pH 5.0 is loaded on the cation exchange material.
  • a conditioned Q-SEPHAROSE® pool, pH 5.5 is loaded onto the cation exchange material.
  • Exemplary load densities are in the range from about 10 to about 100 g/L resin, preferably from about 10 to about 60 g/L resin, most preferably from about 15 to about 45 g/L resin.
  • the antibody of interest is bound to the cation exchange material as a result of this loading step.
  • the cation exchange material is washed in a first wash step with a first wash buffer.
  • wash buffer is passed over the cation exchange material.
  • the composition of the wash buffer is typically chosen to elute as many contaminants as possible from the resin without eluting a substantial amount of the antibody of interest.
  • the pH of the first wash buffer is generally higher than that of the equilibration buffer and/or of the loaded composition, for example about 2 to about 3 pH units higher.
  • the pH of the first wash buffer is in the range from about pH 6.8 to about 9.0, preferably from about pH 6.8 to about 8.0, for example about pH 7.8 or about pH 7.0.
  • buffers which buffer in this pH range include, but are not limited to HEPES, MES, sodium acetate, TRIS/HCl, Triethanolamine hydrochloride/NaOH, Bicine/HCl, Tricine/HCl etc.
  • the preferred first wash buffer comprises or consists of: (1) 25 mM HEPES, pH 7.8 or (2) 25 mM MOPS, pH 7.0.
  • the present invention provides a composition comprising a recombinant chimeric CD20 antibody, such as rituximab, in 25 mM HEPES, pH 7.8.
  • a recombinant humanized VEGF antibody such as bevacizumab, in 25 mM MOPS, pH 7.0.
  • Such compositions are useful, among other things, as intermediate compositions used in the purification of these products.
  • the invention herein generally entails at least one further, or a second, wash step using a second wash buffer.
  • the pH of the second wash buffer preferably is lower than that of the first wash buffer, for example from about 2 to about 3 pH units lower. So, for example, the pH of the second wash buffer may be in the range from about pH 5.0 to about pH 6.0. Preferably, the pH of the second wash buffer is about 5.5.
  • buffers which buffer in this pH range include, but are not limited to, MES, acetic acid/sodium acetate or NaOH, NaH 2 PO 3 /Na 2 HPO 4 , Bis.Tris/HCl. MES, pH 5.5 is the preferred buffer for the second wash.
  • the second wash buffer comprises or consists of: 19 mM MES, 10 mM NaCl, pH 5.50. In another embodiment, the second wash buffer comprises or consists of 23 mM MES, 10 mM NaCl, pH 5.50.
  • first and second wash steps may be employed, preferably only a first and second wash step are performed, prior to eluting the desired antibody.
  • Contaminants such as those discussed above are removed from the cation exchange material during the first and/or second wash step.
  • the first wash step removes most of the contaminants.
  • the desired antibody is eluted from the cation exchange material. Elution of the antibody may be achieved by increasing the conductivity or ionic strength. Desirably, the conductivity of the elution buffer is greater than about 10 mS/cm. Increased conductivity may be achieved by including a relatively high salt concentration in the elution buffer. Exemplary salts for this purpose include, without limitation, sodium acetate, sodium chloride (NaCl), and potassium chloride (KCl). In one embodiment, the elution buffer comprises from about 100 to about 300 mM NaCl. The elution buffer generally will have approximately the same pH as the second wash buffer.
  • a preferred elution buffer comprises: 19 mM MES, 160 mM NaCl, pH 5.5.
  • Another preferred elution buffer comprises: 23 mM MES, 175 mM NaCl, pH 5.5.
  • Elution preferably involves step elution (as opposed to gradient elution).
  • elution step is optionally followed by a regeneration step, such is not necessary according to the preferred embodiment of the invention.
  • the cation exchange purification method herein consists of only the following steps: equilibration (e.g. using equilibration buffer pH about 5.5), loading a composition comprising antibody and contaminant(s) (e.g. where pH of the loaded composition is about 5.0 or about 5.5), first wash step for eluting contaminants (e.g. using first wash buffer pH about 7.8 or first wash buffer pH about 7.0), second wash step (e.g. using second wash buffer pH about 5.5), and elution (e.g. using elution buffer pH about 5.5, and increased conductivity relative to each of the earlier steps for eluting antibody).
  • equilibration e.g. using equilibration buffer pH about 5.5
  • loading a composition comprising antibody and contaminant(s) e.g. where pH of the loaded composition is about 5.0 or about 5.5
  • first wash step for eluting contaminants e.g. using first wash buffer pH about 7.8 or first wash buffer pH about 7.0
  • the antibody preparation obtained according to the cation exchange chromatography method herein may be subjected to additional purification steps, if necessary. Exemplary further purification steps have been discussed above.
  • the antibody is conjugated to one or more heterologous molecules as desired.
  • the heterologous molecule may, for example, be one which increases the serum half-life of the antibody (e.g. polyethylene glycol, PEG), or it may be a label (e.g. an enzyme, fluorescent label and/or radionuclide), or a cytotoxic molecule (e.g. a toxin, chemotherapeutic drug, or radioactive isotope etc).
  • a therapeutic formulation comprising the antibody, optionally conjugated with a heterologous molecule, may be prepared by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • “Pharmaceutically acceptable” carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
  • the active ingredients may also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • the formulation to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody variant, which matrices are in the form of shaped articles, e.g., films, or microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-( ⁇ )-3-hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-( ⁇ )-3-hydroxybutyric acid poly-D-( ⁇ )-3-hydroxybutyric acid.
  • the antibody purified as disclosed herein or the composition comprising the antibody and a pharmaceutically acceptable carrier is then used for various diagnostic, therapeutic or other uses known for such antibodies and compositions.
  • the antibody may be used to treat a disorder in a mammal by administering a therapeutically effective amount of the antibody to the mammal.
  • a CD20 antibody such as rituximab it can be used to deplete B-cells, treat lymphoma (for example Non-Hodgkin's Lymphoma, NHL), or leukemia (for example Chronic Lymphocytic Leukemia, CLL) as well as autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), lupus etc.
  • VEGF for an antibody that binds to VEGF, such as bevacizumab, it can be used to inhibit angiogenesis, treat cancer, and treat macular degeneration, etc.
  • This example describes an improved cation exchange chromatography process for purifying a CD20 antibody, rituximab.
  • Rituximab is used for therapy of NHL, CLL, RA, MS, etc.
  • the structure of the Rituximab molecule is disclosed in U.S. Pat. No. 5,736,137, Anderson et al., (expressly incorporated herein by reference) as well as FIGS. 1A-1B herein.
  • Rituximab is commercially available from Genentech, Inc.
  • Cation-exchange chromatography is used to further reduce the levels of CHOP, DNA, leached protein A, garamycin (GENTAMYCIN®), Rituximab aggregates, and potential viruses.
  • Rituximab binds to the column under the load conditions. The column is then washed, eluted, regenerated/sanitized, and stored until the next use. Multiple cycles may be used to process an entire batch of affinity pool.
  • the cation-exchange pool may be held at room temperature up to 30° C. for up to 3 days or at 5° C. for up to 7 days.
  • the cation-exchange resin (POROS 50 HS®, Applied Biosystems) is packed in a column to a bed height of 17-33 cm. Before the affinity pool is loaded, the cation-exchange column is purged of storage solution with equilibration buffer. After equilibration, the affinity pool is loaded onto the column. The product binds to the column under these conditions. The column is then washed with wash 1 buffer, followed by wash 2 buffer. Rituximab is eluted from the column using a high-ionic-strength elution buffer.
  • Buffer composition Buffer composition Phase (original process) (exemplified process) Pre-equilibration 20 mM MES, 500 mM NaCl, None pH 5.50 Equilibration 20 mM MES, 60 mM NaCl, 19 mM MES, 60 mM NaCl, pH 5.50 pH 5.50 Load Conditioned Protein A pool, Conditioned Protein A pool, pH 5.00, Load density ⁇ 50 g/L pH 5.00, Load density ⁇ 50 g/L resin resin Wash 1 20 mM MES, 60 mM NaCl, 25 mM HEPES, pH 7.80 pH 5.50 Wash 2 None 19 mM MES, 10 mM NaCl, pH 5.50 Elution 20 mM MES, 160 mM NaCl, 19 mM MES, 160 mM NaCl, pH 5.50 pH 5.50 Regeneration 20 mM MES, 500 mM NaCl, None pH 5.50 Equilibration 20 mM MES, 60
  • FIG. 3 illustrates the advantages of the present process in terms of host cell proteins removal.
  • This example describes a cation exchange chromatography process for purifying a recombinant humanized vascular endothelial growth factor antibody (rhuMAb VEGF), bevacizumab.
  • rhuMAb VEGF humanized vascular endothelial growth factor antibody
  • bevacizumab The structure of the bevacizumab molecule is disclosed in U.S. Pat. No. 7,169,901, Presta et al., expressly incorporated herein by reference. See also FIGS. 2A-2B herein. Bevacizumab is commercially available from Genentech, Inc.
  • CM SEPHAROSE FAST FLOW® CM SEPHAROSE FAST FLOW®
  • SP SEPHAROSE FAST FLOW® SP SEPHAROSE FAST FLOW®
  • POROS 50HS® The cation exchange purification processes using these three resins were evaluated with respect to: process performance (impurities removal, retrovirus removal, and step yield), product quality, process robustness and process fit at all current manufacturing sites. Based on the data generated in these studies, POROS 50HS® showed superior process performance and robustness and was selected as the cation exchange resin for the improved purification process.
  • Cation exchange chromatography is the final chromatography step in the purification process. It serves to remove cell culture media components (garamycin), host cell derived impurities (CHOP, and DNA) and aggregated forms of bevacizumab. It also functions as a viral removal step.
  • the column is operated in a bind-and-elute mode and is performed at ambient temperature.
  • the column uses a cation exchange resin (POROS 50HS®).
  • the resin consists of a porous, polystyrene-divinylbenzene bed support coupled with a negatively charged functional group.
  • the column is removed from storage by washing with equilibration buffer.
  • the viral filtered pool will be diluted with 0.3 volumes of water for injection (WFI) to meet the conductivity limit of ⁇ 5.5 mS/cm.
  • WFI water for injection
  • the viral filtered pool is then loaded onto the equilibrated column.
  • the product binds to the resin.
  • the column is washed with a high pH buffer to flush the load material through the column and remove CHOP impurities.
  • the column is then washed with a low salt buffer to lower the pH and prepare the column for elution.
  • Product is eluted using a step elution of high salt buffer with a maximum of 7 column volumes.
  • the column and skid are sanitized with sanitization solution (0.5 N NaOH) prior to storage in storage solution (0.1 N NaOH) until its next use.
  • the desired pH, conductivity and molarity ranges for the load and buffers in the bevacizumab process are provided in the following table.
  • the present process was found to be superior to the original bevacizumab process which used a first wash buffer pH 5.5.
  • the new process herein was able to achieve pools with lower CHOP levels, it achieved a higher step yield and was an overall more robust process to run in manufacturing.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Rheumatology (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Pain & Pain Management (AREA)
  • Vascular Medicine (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Neurology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
US12/260,623 2007-10-30 2008-10-29 Antibody purification by cation exchange chromatography Abandoned US20090148435A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US12/260,623 US20090148435A1 (en) 2007-10-30 2008-10-29 Antibody purification by cation exchange chromatography
US14/531,880 US9896478B2 (en) 2007-10-30 2014-11-03 Antibody purification by cation exchange chromatography
US15/850,885 US20180118781A1 (en) 2007-10-30 2017-12-21 Antibody purification by cation exchange chromatography

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US98382507P 2007-10-30 2007-10-30
US12/260,623 US20090148435A1 (en) 2007-10-30 2008-10-29 Antibody purification by cation exchange chromatography

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/531,880 Continuation US9896478B2 (en) 2007-10-30 2014-11-03 Antibody purification by cation exchange chromatography

Publications (1)

Publication Number Publication Date
US20090148435A1 true US20090148435A1 (en) 2009-06-11

Family

ID=40262967

Family Applications (3)

Application Number Title Priority Date Filing Date
US12/260,623 Abandoned US20090148435A1 (en) 2007-10-30 2008-10-29 Antibody purification by cation exchange chromatography
US14/531,880 Active 2029-09-09 US9896478B2 (en) 2007-10-30 2014-11-03 Antibody purification by cation exchange chromatography
US15/850,885 Abandoned US20180118781A1 (en) 2007-10-30 2017-12-21 Antibody purification by cation exchange chromatography

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14/531,880 Active 2029-09-09 US9896478B2 (en) 2007-10-30 2014-11-03 Antibody purification by cation exchange chromatography
US15/850,885 Abandoned US20180118781A1 (en) 2007-10-30 2017-12-21 Antibody purification by cation exchange chromatography

Country Status (32)

Country Link
US (3) US20090148435A1 (ja)
EP (4) EP3441402A1 (ja)
JP (5) JP5237382B2 (ja)
KR (3) KR101241486B1 (ja)
CN (3) CN103554215B (ja)
AR (2) AR069097A1 (ja)
AU (1) AU2008318865B2 (ja)
BR (1) BRPI0817182A2 (ja)
CA (1) CA2703279C (ja)
CL (1) CL2008003218A1 (ja)
CO (1) CO6280422A2 (ja)
CY (1) CY1116129T1 (ja)
DK (3) DK2565206T3 (ja)
ES (3) ES2666170T3 (ja)
HK (4) HK1143821A1 (ja)
HR (2) HRP20150282T4 (ja)
HU (3) HUE024877T2 (ja)
IL (4) IL205310A0 (ja)
ME (1) ME02101B (ja)
MX (1) MX2010004740A (ja)
NZ (1) NZ584839A (ja)
PE (1) PE20091434A1 (ja)
PH (1) PH12013501128A1 (ja)
PL (3) PL2840090T3 (ja)
PT (1) PT2215117E (ja)
RS (1) RS53850B2 (ja)
RU (1) RU2498991C2 (ja)
SG (2) SG175597A1 (ja)
SI (3) SI2215117T2 (ja)
TW (2) TWI448330B (ja)
WO (1) WO2009058812A1 (ja)
ZA (2) ZA201002850B (ja)

Cited By (38)

* 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
US20090081223A1 (en) * 2005-01-21 2009-03-26 Genentech, Inc. Fixed dosing of her antibodies
US20110076273A1 (en) * 2009-09-11 2011-03-31 Genentech, Inc. Highly Concentrated Pharmaceutical Formulations
WO2011123507A1 (en) * 2010-03-30 2011-10-06 Centocor Ortho Biotech Inc. Humanized il-25 antibodies
WO2011150110A1 (en) * 2010-05-25 2011-12-01 Genentech, Inc. Methods of purifying polypeptides
US20120178910A1 (en) * 2009-09-23 2012-07-12 Medarex, Inc. Cation exchange chromatography (methods)
WO2013055874A2 (en) 2011-10-14 2013-04-18 Genentech, Inc. Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
US20140018525A1 (en) * 2011-03-29 2014-01-16 Glaxosmithkline Llc Buffer system for protein purification
US8691232B2 (en) 2005-02-23 2014-04-08 Genentech, Inc. Extending time to disease progression or survival in cancer patients
US8940302B2 (en) 2007-03-02 2015-01-27 Genentech, Inc. Predicting response to a HER inhibitor
US9017671B2 (en) 2004-10-20 2015-04-28 Genentech, Inc. Method of treating cancer with a pharmaceutical formulation comprising a HER2 antibody
US20150218208A1 (en) * 2012-08-27 2015-08-06 Asahi Kasei Medical Co., Ltd. Method for purifying antibody by temperature-responsive chromatography
WO2015164665A1 (en) 2014-04-25 2015-10-29 Genentech, Inc. Methods of treating early breast cancer with trastuzumab-mcc-dm1 and pertuzumab
US9181346B2 (en) 2008-01-30 2015-11-10 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
KR101657690B1 (ko) * 2015-06-05 2016-09-19 주식회사 녹십자홀딩스 혈장 유래 b형 간염 사람 면역글로불린 제제의 제조방법
WO2016196373A2 (en) 2015-05-30 2016-12-08 Genentech, Inc. Methods of treating her2-positive metastatic breast cancer
WO2017087280A1 (en) 2015-11-16 2017-05-26 Genentech, Inc. Methods of treating her2-positive cancer
US9815904B2 (en) 2013-04-16 2017-11-14 Genetech, Inc. Pertuzumab variants and evaluation thereof
US9896478B2 (en) 2007-10-30 2018-02-20 Genentech, Inc. Antibody purification by cation exchange chromatography
WO2018125589A1 (en) 2016-12-28 2018-07-05 Genentech, Inc. Treatment of advanced her2 expressing cancer
WO2018136412A2 (en) 2017-01-17 2018-07-26 Genentech, Inc. Subcutaneous her2 antibody formulations
WO2018160654A2 (en) 2017-03-02 2018-09-07 Genentech, Inc. Adjuvant treatment of her2-positive breast cancer
WO2018200505A1 (en) 2017-04-24 2018-11-01 Genentech, Inc. Erbb2/her2 mutations in the transmbrane or juxtamembrane domain
WO2018237159A1 (en) * 2017-06-21 2018-12-27 Cephalon, Inc. WASH PAD FOR CATIONIC EXCHANGE CHROMATOGRAPHY
US10246484B2 (en) * 2013-11-06 2019-04-02 Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. Method for purifying recombinant protein
US10532099B2 (en) 2016-10-18 2020-01-14 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
US20200055923A1 (en) * 2016-10-31 2020-02-20 Hexal Ag Antibody Preparation
US10689457B2 (en) 2008-06-16 2020-06-23 Genentech, Inc. Treatment of metastatic breast cancer
US10688164B2 (en) 2015-11-20 2020-06-23 Oregon Health & Science University CMV vectors comprising microRNA recognition elements
US10760097B2 (en) 2011-06-10 2020-09-01 Oregon Health & Science University CMV glycoproteins and recombinant vectors
CN112105927A (zh) * 2018-05-08 2020-12-18 沃特世科技公司 可用于pH梯度阳离子交换色谱法的方法、组合物和试剂盒
US10995121B2 (en) 2014-07-16 2021-05-04 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US11266732B2 (en) 2010-05-14 2022-03-08 Oregon Health & Science University Recombinant HCMV and RHCMV vectors and uses thereof
CN114599971A (zh) * 2019-10-14 2022-06-07 皮尔斯生物科技有限公司 肽纯化配制物和方法
US11416468B2 (en) * 2020-07-21 2022-08-16 International Business Machines Corporation Active-active system index management
CN115850493A (zh) * 2022-11-08 2023-03-28 江苏耀海生物制药有限公司 一种二价纳米抗体Cablivi的分离纯化方法
US11945837B2 (en) 2011-12-22 2024-04-02 Genentech, Inc. Ion exchange membrane chromatography
US12128103B2 (en) 2024-04-16 2024-10-29 Genentech, Inc. Adjuvant treatment of HER2-positive breast cancer

Families Citing this family (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8911964B2 (en) 2006-09-13 2014-12-16 Abbvie Inc. Fed-batch method of making human anti-TNF-alpha antibody
SG10201510384UA (en) 2006-09-13 2016-01-28 Abbvie Inc Cell culture improvements
TW201014605A (en) 2008-09-16 2010-04-16 Genentech Inc Methods for treating progressive multiple sclerosis
US8268314B2 (en) 2008-10-08 2012-09-18 Hoffmann-La Roche Inc. Bispecific anti-VEGF/anti-ANG-2 antibodies
WO2010048192A2 (en) 2008-10-20 2010-04-29 Abbott Laboratories Viral inactivation during purification of antibodies
MX2011004200A (es) 2008-10-20 2011-05-24 Abbott Lab Aislamento y purificacion de anticuerpos usando la cromatografia de afinidad de proteina a.
RU2012140447A (ru) * 2010-02-23 2014-03-27 Дженентек, Инк. Антиангиогенная терапия для лечения рака яичника
AR080794A1 (es) * 2010-03-26 2012-05-09 Hoffmann La Roche Anticuerpos bivalentes biespecificos anti- vegf/ anti-ang-2
GB201012603D0 (en) 2010-07-27 2010-09-08 Ucb Pharma Sa Protein purification
JP5838221B2 (ja) * 2010-12-21 2016-01-06 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト アイソフォーム濃縮抗体調製物及びこれを得る方法
US20140107321A1 (en) * 2011-05-26 2014-04-17 Dr. Reddy's Laboratories Limited Purification of antibodies
WO2013054250A1 (en) * 2011-10-10 2013-04-18 Dr Reddy's Laboratories Limited Purification method
RU2478646C1 (ru) * 2011-11-28 2013-04-10 Федеральное государственное бюджетное учреждение науки Институт молекулярной биологии им.В.А.Энгельгардта Российской академии наук (ИМБ РАН) Способ получения высокоаффинных поликлональных антител
RS62509B1 (sr) 2012-07-13 2021-11-30 Roche Glycart Ag Bispecifična anti-vegf/anti-ang-2 antitela i njihova upotreba u lečenju očnih vaskularnih bolesti
CN103217499B (zh) * 2013-01-15 2015-12-02 珠海市丽珠单抗生物技术有限公司 一种测定免疫球蛋白电荷异构体的糖基化和末端修饰情况的方法
SG10201709131UA (en) 2013-03-08 2017-12-28 Genzyme Corp Continuous purification of therapeutic proteins
UY35517A (es) 2013-04-04 2014-10-31 Mabxience S A Un procedimiento para aumentar la formación de ácido piroglutamico de una proteína
WO2015064971A1 (ko) * 2013-10-30 2015-05-07 (주)셀트리온 양이온 교환 크로마토그래피를 이용한 항체의 아형 분리 방법
CN105979963B (zh) * 2013-11-25 2020-03-03 西雅图基因公司 从cho细胞培养物中制备抗体用于偶联
TWI709569B (zh) 2014-01-17 2020-11-11 美商健臻公司 無菌層析樹脂及其用於製造方法的用途
TWI709570B (zh) 2014-01-17 2020-11-11 美商健臻公司 無菌層析法及製法
EP3102589A1 (en) 2014-02-04 2016-12-14 Biogen MA Inc. Use of cation-exchange chromatography in the flow-through mode to enrich post-translational modifications
PL3116891T3 (pl) 2014-03-10 2020-07-27 Richter Gedeon Nyrt. Oczyszczanie immunoglobuliny z zastosowaniem etapów wstępnego oczyszczania
CN105017418B (zh) * 2014-03-27 2021-02-23 上海药明康德新药开发有限公司 单克隆抗体纯化工艺方法
CN105315369B (zh) * 2014-07-25 2020-03-13 山东博安生物技术有限公司 利用阳离子交换层析纯化蛋白质
EP3268100A1 (en) * 2015-03-13 2018-01-17 Bristol-Myers Squibb Company Use of alkaline washes during chromatography to remove impurities
GB201600512D0 (en) * 2016-01-12 2016-02-24 Univ York Recombinant protein production
EP3452103A1 (en) 2016-04-15 2019-03-13 The Trustees Of The University Of Pennsylvania Compositions for treatment of wet age-related macular degeneration
US10654922B2 (en) * 2016-05-13 2020-05-19 Askgene Pharma Inc. Angiopoietin 2, VEGF dual antagonists
EP3484911B1 (en) * 2016-07-15 2020-11-04 H. Hoffnabb-La Roche Ag Method for purifying pegylated erythropoietin
CN106222222B (zh) * 2016-08-08 2019-10-29 湖北医药学院 一种重组人白血病抑制因子的制备方法
WO2018045587A1 (zh) * 2016-09-12 2018-03-15 广东东阳光药业有限公司 一种抗vegf类单克隆抗体的纯化方法
CN106380519B (zh) * 2016-10-17 2019-11-01 深圳万乐药业有限公司 一种单克隆抗体的纯化方法
BE1025090B1 (fr) 2017-03-30 2018-10-29 Univercells Sa Procede et kit de purification de proteines
TWI679209B (zh) * 2017-04-14 2019-12-11 南韓商Ｃｊ醫藥健康股份有限公司 使用陽離子交換層析法純化同功抗體之方法
WO2019060718A1 (en) * 2017-09-22 2019-03-28 Immunogen, Inc. SEPARATION OF LIGHT TRIPLE CHAIN ANTIBODIES USING CATION EXCHANGE CHROMATOGRAPHY
CN110272491B (zh) * 2018-03-13 2023-01-24 江苏恒瑞医药股份有限公司 一种抗pd-1抗体的纯化工艺
KR20200136464A (ko) * 2018-03-29 2020-12-07 브리스톨-마이어스 스큅 컴퍼니 단량체성 모노클로날 항체를 정제하는 방법
CA3110666A1 (en) 2018-08-31 2020-03-05 Genzyme Corporation Sterile chromatography resin and use thereof in manufacturing processes
EP3643322A1 (en) * 2018-10-26 2020-04-29 Mabion SA Low aggregate anti cd20 ligand formulation
CN109320611B (zh) * 2018-10-31 2022-06-03 鼎康(武汉)生物医药有限公司 一种人鼠嵌合单克隆抗体生物类似药的纯化方法
CN112206327A (zh) * 2019-07-12 2021-01-12 上海药明生物技术有限公司 一种抗体偶联药物的制备及其高通量筛选方法
US20230027029A1 (en) * 2019-11-25 2023-01-26 Akeso Biopharma, Inc. Anti-pd-1-anti-vegfa bispecific antibody, pharmaceutical composition and use thereof
KR102153258B1 (ko) * 2020-02-21 2020-09-07 프레스티지바이오로직스 주식회사 베바시주맙 정제의 최적화된 방법
CN114591438B (zh) * 2022-04-25 2023-12-05 达石药业(广东)有限公司 一种采用阳离子交换层析法纯化双特异性抗体的方法
WO2024090489A1 (ja) * 2022-10-26 2024-05-02 日本メジフィジックス株式会社 放射性医薬組成物の製造方法
CN117756878A (zh) * 2023-12-26 2024-03-26 康日百奥生物科技(苏州)有限公司 抗体层析分离方法及应用

Citations (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4753894A (en) * 1984-02-08 1988-06-28 Cetus Corporation Monoclonal anti-human breast cancer antibodies
US4966851A (en) * 1986-12-01 1990-10-30 The University Of British Columbia Process for isolation of lysozyme and avidin from egg white
US5110913A (en) * 1990-05-25 1992-05-05 Miles Inc. Antibody purification method
US5112951A (en) * 1989-07-28 1992-05-12 Hybritech Incorporated Separation of anti-metal chelate antibodies
US5115101A (en) * 1988-06-08 1992-05-19 Miles Inc. Removal of protein A from antibody preparations
US5118796A (en) * 1987-12-09 1992-06-02 Centocor, Incorporated Efficient large-scale purification of immunoglobulins and derivatives
US5169774A (en) * 1984-02-08 1992-12-08 Cetus Oncology Corporation Monoclonal anti-human breast cancer antibodies
US5196323A (en) * 1985-04-27 1993-03-23 Boehringer Ingelheim International Gmbh Process for preparing and purifying alpha-interferon
US5256769A (en) * 1984-09-26 1993-10-26 Takeda Chemical Industries, Ltd. Mutual separation of proteins
US5279823A (en) * 1992-06-08 1994-01-18 Genentech, Inc. Purified forms of DNASE
US5429746A (en) * 1994-02-22 1995-07-04 Smith Kline Beecham Corporation Antibody purification
US5451662A (en) * 1987-10-23 1995-09-19 Schering Corporation Method of purifying protein
US5525338A (en) * 1992-08-21 1996-06-11 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin conjugates
US5677171A (en) * 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
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
US6005081A (en) * 1996-11-15 1999-12-21 Genentech, Inc. Purification of recombinant human neurotrophins
US6054561A (en) * 1984-02-08 2000-04-25 Chiron Corporation Antigen-binding sites of antibody molecules specific for cancer antigens
US6127526A (en) * 1996-11-27 2000-10-03 Genentech, Inc. Protein purification by Protein A chromatography
US6267958B1 (en) * 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6339142B1 (en) * 1998-05-06 2002-01-15 Genentech, Inc. Protein purification
US6417355B1 (en) * 2001-04-11 2002-07-09 The United States Of America As Represented By The Secretary Of The Navy Geminal-dinitro-1-5 diazocine derivatives
US20040082047A1 (en) * 2002-09-11 2004-04-29 Emery Jefferson C. Protein purification
US7169901B2 (en) * 1997-04-07 2007-01-30 Genentech, Inc. Anti-VEGF antibodies

Family Cites Families (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3773919A (en) 1969-10-23 1973-11-20 Du Pont Polylactide-drug mixtures
USRE30985E (en) 1978-01-01 1982-06-29 Serum-free cell culture media
US4515893A (en) 1979-04-26 1985-05-07 Ortho Pharmaceutical Corporation Hybrid cell line for producing complement-fixing monoclonal antibody to human T cells
GB2070818A (en) 1980-02-04 1981-09-09 Philips Electronic Associated Regulated power supply for an image intensifier
WO1984002129A1 (en) 1982-11-22 1984-06-07 Takeda Chemical Industries Ltd Human immune interferon protein and process for its preparation
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
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
DD266710A3 (de) 1983-06-06 1989-04-12 Ve Forschungszentrum Biotechnologie Verfahren zur biotechnischen Herstellung van alkalischer Phosphatase
US4767704A (en) 1983-10-07 1988-08-30 Columbia University In The City Of New York Protein-free culture medium
US4879231A (en) 1984-10-30 1989-11-07 Phillips Petroleum Company Transformation of yeasts of the genus pichia
GB8516415D0 (en) 1985-06-28 1985-07-31 Celltech Ltd Culture of animal cells
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
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
IT1219874B (it) 1988-03-18 1990-05-24 Fidia Farmaceutici Utilizzazione del fattore di crescita nervoso umano e sue composizioni farmaceutiche
ATE113846T1 (de) 1988-06-21 1994-11-15 Genentech Inc Therapeutische zusammensetzungen für die behandlung von myocard-infarkten.
ATE135397T1 (de) 1988-09-23 1996-03-15 Cetus Oncology Corp Zellenzuchtmedium für erhöhtes zellenwachstum, zur erhöhung der langlebigkeit und expression der produkte
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
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
EP0467466A1 (en) * 1990-07-16 1992-01-22 Eastman Kodak Company Method for the purification of immunoreactive labeled thyroxine conjugates
US5122469A (en) 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins
LU91067I2 (fr) 1991-06-14 2004-04-02 Genentech Inc Trastuzumab et ses variantes et dérivés immuno chimiques y compris les immotoxines
DE122006000006I2 (de) 1991-08-14 2011-06-16 Genentech Inc Veränderte Immunglobuline für spezifische FC-Epsilon Rezeptoren
US7018809B1 (en) 1991-09-19 2006-03-28 Genentech, Inc. Expression of functional antibody fragments
ES2241710T3 (es) 1991-11-25 2005-11-01 Enzon, Inc. Procedimiento para producir proteinas multivalentes de union a antigeno.
JPH05202098A (ja) 1992-01-29 1993-08-10 Snow Brand Milk Prod Co Ltd 乳質原料から生理活性物質の製造法
CA2372813A1 (en) 1992-02-06 1993-08-19 L.L. Houston Biosynthetic binding protein for cancer marker
AU687755B2 (en) 1992-08-21 1998-03-05 Genentech Inc. Method for treating an LFA-1-mediated disorder
ATE196606T1 (de) 1992-11-13 2000-10-15 Idec Pharma Corp Therapeutische verwendung von chimerischen und markierten antikörpern, die gegen ein differenzierung-antigen gerichtet sind, dessen expression auf menschliche b lymphozyt beschränkt ist, für die behandlung von b-zell-lymphoma
US5595721A (en) 1993-09-16 1997-01-21 Coulter Pharmaceutical, Inc. Radioimmunotherapy of lymphoma using anti-CD20
EP0733207B1 (en) 1993-12-10 1997-08-27 Genentech, Inc. Methods for diagnosis of allergy and screening of anti-allergy therapeutics
JP3825798B2 (ja) 1994-01-18 2006-09-27 ジェネンテク，インコーポレイテッド ＩｇＥアンタゴニストを用いる寄生虫感染症の治療法
EP0749488A1 (en) 1994-03-03 1996-12-27 Genentech, Inc. Anti-il-8 monoclonal antibodies for treatment of inflammatory disorders
US5534615A (en) 1994-04-25 1996-07-09 Genentech, Inc. Cardiac hypertrophy factor and uses therefor
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
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
JPH11507535A (ja) 1995-06-07 1999-07-06 イムクローン システムズ インコーポレイテッド 腫瘍の成長を抑制する抗体および抗体フラグメント類
US5714583A (en) 1995-06-07 1998-02-03 Genetics Institute, Inc. Factor IX purification methods
DK0877626T3 (da) 1996-01-23 2002-12-30 Univ Vermont Anti-CD18 antistoffer til anvendelse mod slagtilfælde
US7147851B1 (en) 1996-08-15 2006-12-12 Millennium Pharmaceuticals, Inc. Humanized immunoglobulin reactive with α4β7 integrin
KR100532178B1 (ko) 1996-11-27 2005-12-01 제넨테크, 인크. 인간화 항-ＣＤ11ａ 항체
NZ500078A (en) 1997-04-07 2001-10-26 Genentech Inc Humanized anti-VEGF antibodies and their use in inhibiting VEGF-induced angiogenesis in mammals
BRPI9809388B8 (pt) 1997-04-07 2021-05-25 Genentech Inc anticorpos humanizados e métodos para a formação de anticorpos humanizados.
DK1860187T3 (da) 1997-05-15 2011-10-31 Genentech Inc Apo-2-receptor
US5994511A (en) 1997-07-02 1999-11-30 Genentech, Inc. Anti-IgE antibodies and methods of improving polypeptides
ES2228052T3 (es) 1998-06-01 2005-04-01 Genentech, Inc. Separacion demonomeros de anticuerpos de sus multimeros utilizando cromatografia de intercambio de iones.
AU753468B2 (en) 1998-06-09 2002-10-17 Csl Behring Ag Process for producing immunoglobulins for intravenous administration and other immunoglobulin products
IL146954A0 (en) 1999-06-25 2002-08-14 Genentech Inc HUMANIZED ANTI-ErbB2 ANTIBODIES AND TREATMENT WITH ANTI-ErbB2 ANTIBODIES
IL149116A0 (en) 1999-10-29 2002-11-10 Genentech Inc Anti-prostate stem cell antigen (psca) antibody compositions and methods of use
UA83458C2 (uk) 2000-09-18 2008-07-25 Байоджен Айдек Ма Інк. Виділений поліпептид baff-r (рецептор фактора активації в-клітин сімейства tnf)
GB0113179D0 (en) * 2001-05-31 2001-07-25 Novartis Ag Organic compounds
US7321026B2 (en) 2001-06-27 2008-01-22 Skytech Technology Limited Framework-patched immunoglobulins
US6770027B2 (en) * 2001-10-05 2004-08-03 Scimed Life Systems, Inc. Robotic endoscope with wireless interface
WO2003033658A2 (en) 2001-10-17 2003-04-24 Human Genome Sciences, Inc. Neutrokine-alpha and neutrokine-alpha splice variant
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
AU2003208415B2 (en) 2002-02-14 2009-05-28 Immunomedics, Inc. Anti-CD20 antibodies and fusion proteins thereof and methods of use
US20060182740A1 (en) * 2002-06-21 2006-08-17 Biogen Idec, Inc. Buffered formulations for concentrating antibodies and methods of use thereof
PL218660B1 (pl) 2002-10-17 2015-01-30 Genmab As Izolowane ludzkie przeciwciało monoklonalne wiążące ludzki CD20, związane z tym przeciwciałem transfektoma, komórka gospodarza, transgeniczne zwierzę lub roślina, kompozycja, immunokoniugat, cząsteczka bispecyficzna, wektor ekspresyjny, kompozycja farmaceutyczna, zastosowanie medyczne, zestaw oraz przeciwciało antyidiotypowe i jego zastosowanie
PL377213A1 (pl) 2002-12-13 2006-01-23 Mitra Medical Technology Ab Antychłoniakowe środki ukierunkowujące z funkcjami efektorowymi i powinowactwa połączonymi trifunkcyjnym reagentem
EP2301966A1 (en) 2002-12-16 2011-03-30 Genentech, Inc. Immunoglobulin variants and uses thereof
ZA200505306B (en) 2002-12-31 2006-09-27 Altus Pharmaceuticals Inc Complexes of protein crystals and ionic polymers
WO2004087761A1 (ja) * 2003-03-31 2004-10-14 Kirin Beer Kabushiki Kaisha ヒトモノクローナル抗体およびヒトポリクローナル抗体の精製
KR101412271B1 (ko) 2003-05-09 2014-06-25 듀크 유니버시티 Ｃｄ２０-특이적 항체 및 이를 이용한 방법
AR044388A1 (es) 2003-05-20 2005-09-07 Applied Molecular Evolution Moleculas de union a cd20
AU2004263538B2 (en) 2003-08-08 2009-09-17 Immunomedics, Inc. Bispecific antibodies for inducing apoptosis of tumor and diseased cells
US8147832B2 (en) 2003-08-14 2012-04-03 Merck Patent Gmbh CD20-binding polypeptide compositions and methods
KR100524074B1 (ko) 2003-10-01 2005-10-26 삼성전자주식회사 베젤 구조를 가지는 전자기기
TW200533357A (en) † 2004-01-08 2005-10-16 Millennium Pharm Inc 2-(amino-substituted)-4-aryl pyrimidines and related compounds useful for treating inflammatory diseases
BRPI0509412A (pt) * 2004-04-16 2007-09-04 Genentech Inc método de tratamento de policondrite ou mononeurite multiplex em mamìferos e artigo industrializado
CN101022829A (zh) * 2004-04-16 2007-08-22 健泰科生物技术公司 用抗cd20抗体治疗多软骨炎和多发性单神经炎
JP2008525041A (ja) * 2004-12-22 2008-07-17 ジェネンテック・インコーポレーテッド 可溶性多膜貫通型タンパク質の産生方法
ES2797480T3 (es) * 2005-03-11 2020-12-02 Wyeth Llc Un procedimiento de cromatografía de reparto débil
AR053633A1 (es) * 2005-06-17 2007-05-09 Wyeth Corp Metodos para purificar proteinas que contienen una region fc
WO2007028154A2 (en) * 2005-09-02 2007-03-08 Northwestern University Encapsulated arsenic drugs
KR20080111487A (ko) * 2006-03-20 2008-12-23 메다렉스, 인코포레이티드 단백질 정제 방법
KR20150006085A (ko) * 2006-04-05 2015-01-15 애브비 바이오테크놀로지 리미티드 항체 정제
MX2009002014A (es) * 2006-08-28 2009-03-09 Ares Trading Sa Proceso para la purificacion de proteinas que contienen fc.
US8620738B2 (en) 2006-08-31 2013-12-31 Visa U.S.A. Inc Loyalty program incentive determination
US20110130544A1 (en) * 2007-03-30 2011-06-02 Medimmune, Llc Antibodies with decreased deamidation profiles
SI2215117T2 (en) * 2007-10-30 2018-04-30 Genentech, Inc. Purification of the antibody by cation exchange chromatography
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

Patent Citations (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6054561A (en) * 1984-02-08 2000-04-25 Chiron Corporation Antigen-binding sites of antibody molecules specific for cancer antigens
US4753894A (en) * 1984-02-08 1988-06-28 Cetus Corporation Monoclonal anti-human breast cancer antibodies
US5169774A (en) * 1984-02-08 1992-12-08 Cetus Oncology Corporation Monoclonal anti-human breast cancer antibodies
US5256769A (en) * 1984-09-26 1993-10-26 Takeda Chemical Industries, Ltd. Mutual separation of proteins
US5196323A (en) * 1985-04-27 1993-03-23 Boehringer Ingelheim International Gmbh Process for preparing and purifying alpha-interferon
US4966851A (en) * 1986-12-01 1990-10-30 The University Of British Columbia Process for isolation of lysozyme and avidin from egg white
US5451662A (en) * 1987-10-23 1995-09-19 Schering Corporation Method of purifying protein
US5118796A (en) * 1987-12-09 1992-06-02 Centocor, Incorporated Efficient large-scale purification of immunoglobulins and derivatives
US5677171A (en) * 1988-01-12 1997-10-14 Genentech, Inc. Monoclonal antibodies directed to the HER2 receptor
US5115101A (en) * 1988-06-08 1992-05-19 Miles Inc. Removal of protein A from antibody preparations
US5112951A (en) * 1989-07-28 1992-05-12 Hybritech Incorporated Separation of anti-metal chelate antibodies
US5110913A (en) * 1990-05-25 1992-05-05 Miles Inc. Antibody purification method
US5279823A (en) * 1992-06-08 1994-01-18 Genentech, Inc. Purified forms of DNASE
US5525338A (en) * 1992-08-21 1996-06-11 Immunomedics, Inc. Detection and therapy of lesions with biotin/avidin conjugates
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
US5429746A (en) * 1994-02-22 1995-07-04 Smith Kline Beecham Corporation Antibody purification
US6267958B1 (en) * 1995-07-27 2001-07-31 Genentech, Inc. Protein formulation
US6005081A (en) * 1996-11-15 1999-12-21 Genentech, Inc. Purification of recombinant human neurotrophins
US6797814B2 (en) * 1996-11-27 2004-09-28 Genentech, Inc. Protein purification
US6127526A (en) * 1996-11-27 2000-10-03 Genentech, Inc. Protein purification by Protein A chromatography
US6333398B1 (en) * 1996-11-27 2001-12-25 Genentech, Inc. Protein purification
US7169901B2 (en) * 1997-04-07 2007-01-30 Genentech, Inc. Anti-VEGF antibodies
US6417335B1 (en) * 1998-05-06 2002-07-09 Genentech, Inc. Protein purification
US6489447B1 (en) * 1998-05-06 2002-12-03 Genentech, Inc. Protein purification
US7074404B2 (en) * 1998-05-06 2006-07-11 Genentech, Inc. Protein purification
US6339142B1 (en) * 1998-05-06 2002-01-15 Genentech, Inc. Protein purification
US6417355B1 (en) * 2001-04-11 2002-07-09 The United States Of America As Represented By The Secretary Of The Navy Geminal-dinitro-1-5 diazocine derivatives
US20040082047A1 (en) * 2002-09-11 2004-04-29 Emery Jefferson C. Protein purification

Cited By (83)

* 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
US8883980B2 (en) 2003-11-05 2014-11-11 Roche Glycart 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
US9017671B2 (en) 2004-10-20 2015-04-28 Genentech, Inc. Method of treating cancer with a pharmaceutical formulation comprising a HER2 antibody
US8404234B2 (en) 2005-01-21 2013-03-26 Genentech, Inc. Fixed dosing of HER antibodies
US20090081223A1 (en) * 2005-01-21 2009-03-26 Genentech, Inc. Fixed dosing of her antibodies
US8691232B2 (en) 2005-02-23 2014-04-08 Genentech, Inc. Extending time to disease progression or survival in cancer patients
US8940302B2 (en) 2007-03-02 2015-01-27 Genentech, Inc. Predicting response to a HER inhibitor
US9896478B2 (en) 2007-10-30 2018-02-20 Genentech, Inc. Antibody purification by cation exchange chromatography
US11597776B2 (en) 2008-01-30 2023-03-07 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US9181346B2 (en) 2008-01-30 2015-11-10 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US11414498B2 (en) 2008-01-30 2022-08-16 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US12110341B2 (en) 2008-01-30 2024-10-08 Genentech, Inc. Composition comprising antibody that binds to domain II of HER2 and acidic variants thereof
US11655305B2 (en) 2008-06-16 2023-05-23 Genentech, Inc. Treatment of metastatic breast cancer
US10689457B2 (en) 2008-06-16 2020-06-23 Genentech, Inc. Treatment of metastatic breast cancer
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
US20190119317A1 (en) * 2009-09-23 2019-04-25 E.R. Squibb & Sons, L.L.C. Cation exchange chromatography methods
US20120178910A1 (en) * 2009-09-23 2012-07-12 Medarex, Inc. Cation exchange chromatography (methods)
US11292814B2 (en) * 2009-09-23 2022-04-05 E.R. Squibb & Sons, L.L.C. Cation exchange chromatography methods
US20220177516A1 (en) * 2009-09-23 2022-06-09 E.R. Squibb & Sons, L.L.C. Cation exchange chromatography methods
US8785605B2 (en) 2010-03-30 2014-07-22 Janssen Biotech, Inc. Humanized IL-25 antibodies
EA031203B1 (ru) * 2010-03-30 2018-11-30 Янссен Байотек Инк. Гуманизированные антитела к il-25
WO2011123507A1 (en) * 2010-03-30 2011-10-06 Centocor Ortho Biotech Inc. Humanized il-25 antibodies
US11266732B2 (en) 2010-05-14 2022-03-08 Oregon Health & Science University Recombinant HCMV and RHCMV vectors and uses thereof
WO2011150110A1 (en) * 2010-05-25 2011-12-01 Genentech, Inc. Methods of purifying polypeptides
JP2013530156A (ja) * 2010-05-25 2013-07-25 ジェネンテック， インコーポレイテッド ポリペプチドの精製方法
KR20130086544A (ko) * 2010-05-25 2013-08-02 제넨테크, 인크. 폴리펩티드의 정제 방법
KR101976853B1 (ko) * 2010-05-25 2019-05-09 제넨테크, 인크. 폴리펩티드의 정제 방법
US20130079272A1 (en) * 2010-05-25 2013-03-28 Genentech, Inc. Methods of purifying polypeptides
US9868761B2 (en) 2011-03-29 2018-01-16 Glaxosmithkline Llc Buffer system for protein purification
AU2012236486B2 (en) * 2011-03-29 2016-06-23 Glaxosmithkline Llc Buffer system for protein purification
US9624261B2 (en) * 2011-03-29 2017-04-18 Glaxosmithkline Llc Buffer system for protein purification
US20140018525A1 (en) * 2011-03-29 2014-01-16 Glaxosmithkline Llc Buffer system for protein purification
US10760097B2 (en) 2011-06-10 2020-09-01 Oregon Health & Science University CMV glycoproteins and recombinant vectors
EP3598981A2 (en) 2011-10-14 2020-01-29 F. Hoffmann-La Roche AG Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
EP4403228A2 (en) 2011-10-14 2024-07-24 F. Hoffmann-La Roche AG Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
WO2013055874A2 (en) 2011-10-14 2013-04-18 Genentech, Inc. Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
EP4234034A2 (en) 2011-10-14 2023-08-30 F. Hoffmann-La Roche AG Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
EP4241849A2 (en) 2011-10-14 2023-09-13 F. Hoffmann-La Roche AG Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
EP4234033A2 (en) 2011-10-14 2023-08-30 F. Hoffmann-La Roche AG Uses for and article of manufacture including her2 dimerization inhibitor pertuzumab
US11945837B2 (en) 2011-12-22 2024-04-02 Genentech, Inc. Ion exchange membrane chromatography
US20150218208A1 (en) * 2012-08-27 2015-08-06 Asahi Kasei Medical Co., Ltd. Method for purifying antibody by temperature-responsive chromatography
US9815904B2 (en) 2013-04-16 2017-11-14 Genetech, Inc. Pertuzumab variants and evaluation thereof
US9969811B2 (en) 2013-04-16 2018-05-15 Genentech, Inc. Pertuzumab variants and evaluation thereof
US10246484B2 (en) * 2013-11-06 2019-04-02 Sunshine Guojian Pharmaceutical (Shanghai) Co., Ltd. Method for purifying recombinant protein
WO2015164665A1 (en) 2014-04-25 2015-10-29 Genentech, Inc. Methods of treating early breast cancer with trastuzumab-mcc-dm1 and pertuzumab
US11692012B2 (en) 2014-07-16 2023-07-04 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
US10995121B2 (en) 2014-07-16 2021-05-04 Oregon Health & Science University Human cytomegalovirus comprising exogenous antigens
WO2016196373A2 (en) 2015-05-30 2016-12-08 Genentech, Inc. Methods of treating her2-positive metastatic breast cancer
US11406715B2 (en) 2015-05-30 2022-08-09 Genentech, Inc. Methods of treating HER2-positive metastatic breast cancer
US20180140699A1 (en) * 2015-06-05 2018-05-24 Green Cross Holdings Corporation Methods for preparing hepatitis b immunoglobulin derived from plasma
CN107849086A (zh) * 2015-06-05 2018-03-27 株式会社绿十字控股 源自血浆的乙型肝炎人免疫球蛋白的制备方法
KR101657690B1 (ko) * 2015-06-05 2016-09-19 주식회사 녹십자홀딩스 혈장 유래 b형 간염 사람 면역글로불린 제제의 제조방법
US10406223B2 (en) * 2015-06-05 2019-09-10 Green Cross Holdings Corporation Methods for preparing hepatitis B immunoglobulin derived from plasma
WO2016195387A1 (ko) * 2015-06-05 2016-12-08 주식회사 녹십자홀딩스 혈장 유래 b형 간염 사람 면역글로불린 제제의 제조방법
WO2017087280A1 (en) 2015-11-16 2017-05-26 Genentech, Inc. Methods of treating her2-positive cancer
US10688164B2 (en) 2015-11-20 2020-06-23 Oregon Health & Science University CMV vectors comprising microRNA recognition elements
US10532099B2 (en) 2016-10-18 2020-01-14 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
US11305015B2 (en) 2016-10-18 2022-04-19 Oregon Health & Science University Cytomegalovirus vectors eliciting T cells restricted by major histocompatibility complex E molecules
US11021530B2 (en) * 2016-10-31 2021-06-01 Hexal Ag Antibody preparation
US20200055923A1 (en) * 2016-10-31 2020-02-20 Hexal Ag Antibody Preparation
WO2018125589A1 (en) 2016-12-28 2018-07-05 Genentech, Inc. Treatment of advanced her2 expressing cancer
US10849849B2 (en) 2017-01-17 2020-12-01 Genentech Inc. Subcutaneous HER2 antibody formulations
WO2018136412A2 (en) 2017-01-17 2018-07-26 Genentech, Inc. Subcutaneous her2 antibody formulations
EP3868404A1 (en) 2017-01-17 2021-08-25 F. Hoffmann-La Roche AG Subcutaneous her2 antibody formulations
US11654105B2 (en) 2017-01-17 2023-05-23 Genentech, Inc. Subcutaneous HER2 antibody formulations
WO2018160654A2 (en) 2017-03-02 2018-09-07 Genentech, Inc. Adjuvant treatment of her2-positive breast cancer
US11992529B2 (en) 2017-03-02 2024-05-28 Genentech, Inc. Adjuvant treatment of HER2-positive breast cancer
US11638756B2 (en) 2017-03-02 2023-05-02 Genentech, Inc. Adjuvant treatment of HER2-positive breast cancer
EP4368199A2 (en) 2017-03-02 2024-05-15 Genentech, Inc. Adjuvant treatment of her2-positive breast cancer
US11077189B2 (en) 2017-03-02 2021-08-03 Genentech Inc. Adjuvant treatment of HER2-positive breast cancer
WO2018200505A1 (en) 2017-04-24 2018-11-01 Genentech, Inc. Erbb2/her2 mutations in the transmbrane or juxtamembrane domain
US11426706B2 (en) 2017-06-21 2022-08-30 Cephalon, Inc. Cation exchange chromatography wash buffer
WO2018237159A1 (en) * 2017-06-21 2018-12-27 Cephalon, Inc. WASH PAD FOR CATIONIC EXCHANGE CHROMATOGRAPHY
CN112105927A (zh) * 2018-05-08 2020-12-18 沃特世科技公司 可用于pH梯度阳离子交换色谱法的方法、组合物和试剂盒
US11612885B2 (en) * 2018-05-08 2023-03-28 Waters Technologies Corporation Methods, compositions and kits useful for pH gradient cation exchange chromatography
CN114599971A (zh) * 2019-10-14 2022-06-07 皮尔斯生物科技有限公司 肽纯化配制物和方法
US11416468B2 (en) * 2020-07-21 2022-08-16 International Business Machines Corporation Active-active system index management
CN115850493A (zh) * 2022-11-08 2023-03-28 江苏耀海生物制药有限公司 一种二价纳米抗体Cablivi的分离纯化方法
US12128103B2 (en) 2024-04-16 2024-10-29 Genentech, Inc. Adjuvant treatment of HER2-positive breast cancer

Also Published As

Publication number Publication date
JP2013173747A (ja) 2013-09-05
RU2498991C2 (ru) 2013-11-20
US20150056196A1 (en) 2015-02-26
HK1221234A1 (zh) 2017-05-26
DK2215117T3 (en) 2015-03-02
IL205310A0 (en) 2010-12-30
ZA201002850B (en) 2011-07-27
SI2840090T1 (en) 2018-05-31
SI2215117T2 (en) 2018-04-30
CN101889025B (zh) 2013-11-13
JP2015145371A (ja) 2015-08-13
PL2215117T5 (pl) 2018-06-29
ES2533266T5 (es) 2018-04-18
IL239495A0 (en) 2015-08-31
US20180118781A1 (en) 2018-05-03
CO6280422A2 (es) 2011-05-20
HK1143821A1 (en) 2011-01-14
CN103554215A (zh) 2014-02-05
ES2572958T3 (es) 2016-06-03
RU2010121816A (ru) 2011-12-10
TWI448330B (zh) 2014-08-11
RS53850B1 (en) 2015-08-31
CA2703279C (en) 2014-04-22
HRP20150282T4 (hr) 2018-08-10
PL2840090T3 (pl) 2018-07-31
RS53850B2 (sr) 2018-07-31
ME02101B (me) 2015-10-20
JP5237382B2 (ja) 2013-07-17
KR101241486B1 (ko) 2013-03-15
SI2565206T1 (sl) 2016-07-29
KR20100086015A (ko) 2010-07-29
HUE027668T2 (en) 2016-11-28
JP5885864B2 (ja) 2016-03-16
ES2533266T3 (es) 2015-04-08
AU2008318865B2 (en) 2012-06-28
SI2215117T1 (sl) 2015-03-31
EP2565206A2 (en) 2013-03-06
EP2215117B2 (en) 2018-01-10
EP2565206A3 (en) 2013-11-06
EP2840090A1 (en) 2015-02-25
PL2565206T3 (pl) 2017-08-31
BRPI0817182A2 (pt) 2015-03-17
PE20091434A1 (es) 2009-10-17
CN103554215B (zh) 2016-06-15
PL2215117T3 (pl) 2015-06-30
AR111171A2 (es) 2019-06-12
CA2703279A1 (en) 2009-05-07
PT2215117E (pt) 2015-04-01
EP2215117B1 (en) 2014-12-31
HK1206753A1 (en) 2016-01-15
IL234901A0 (en) 2014-11-30
IL239495B (en) 2019-09-26
CY1116129T1 (el) 2017-02-08
DK2840090T3 (en) 2018-04-23
EP2565206B1 (en) 2016-03-30
ES2666170T3 (es) 2018-05-03
CN101889025A (zh) 2010-11-17
US9896478B2 (en) 2018-02-20
DK2215117T4 (en) 2018-04-09
HUE024877T2 (en) 2016-02-29
KR20150008503A (ko) 2015-01-22
KR20140015166A (ko) 2014-02-06
DK2565206T3 (en) 2016-06-06
NZ584839A (en) 2012-07-27
JP2015155406A (ja) 2015-08-27
SG10201401690XA (en) 2014-06-27
AR069097A1 (es) 2009-12-30
JP2011502161A (ja) 2011-01-20
AU2008318865A1 (en) 2009-05-07
HRP20180943T1 (hr) 2018-08-10
HK1182401A1 (zh) 2013-11-29
HUE037409T2 (hu) 2018-08-28
JP2017019790A (ja) 2017-01-26
IL234902A0 (en) 2014-11-30
SG175597A1 (en) 2011-11-28
EP3441402A1 (en) 2019-02-13
TW200927289A (en) 2009-07-01
EP2840090B1 (en) 2018-02-21
WO2009058812A1 (en) 2009-05-07
TW201512216A (zh) 2015-04-01
TWI554517B (zh) 2016-10-21
MX2010004740A (es) 2010-05-27
CN105315323A (zh) 2016-02-10
HRP20150282T1 (hr) 2015-06-19
CL2008003218A1 (es) 2009-03-06
PH12013501128A1 (en) 2015-12-02
ZA201102169B (en) 2012-12-27
EP2215117A1 (en) 2010-08-11

Similar Documents

Publication Publication Date Title
US9896478B2 (en) Antibody purification by cation exchange chromatography
EP2321337B1 (en) Methods for removing a contaminant using protein displacement ion exchange membrane chromatography
AU2012227163B2 (en) Antibody purification by cation exchange chromatography
AU2015218432A1 (en) Antibody purification by cation exchange chromatography

Legal Events

Date Code Title Description
AS Assignment

Owner name: GENENTECH, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LEBRETON, BENEDICTE ANDREE;O'CONNOR, DEBORAH ANN;SAFTA, AURELIA;AND OTHERS;REEL/FRAME:022227/0227;SIGNING DATES FROM 20081215 TO 20081216

STCB Information on status: application discontinuation

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