US20090098660A1 - Method for the Purification of Antibodies - Google Patents

Method for the Purification of Antibodies Download PDF

Info

Publication number
US20090098660A1
US20090098660A1 US11920288 US92028806A US2009098660A1 US 20090098660 A1 US20090098660 A1 US 20090098660A1 US 11920288 US11920288 US 11920288 US 92028806 A US92028806 A US 92028806A US 2009098660 A1 US2009098660 A1 US 2009098660A1
Authority
US
Grant status
Application
Patent type
Prior art keywords
step
mm
elution
sodium
concentration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11920288
Inventor
Roberto Falkenstein
Burkard Kolb
Maria Sebald
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.)
Hoffmann-La Roche Inc
Original Assignee
F Hoffmann-La Roche AG
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

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/26Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361Ion-exchange
    • B01D15/362Cation-exchange
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/26Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
    • B01D15/361Ion-exchange
    • B01D15/363Anion-exchange
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01DSEPARATION
    • B01D15/00Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08Selective adsorption, e.g. chromatography
    • B01D15/42Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
    • B01D15/424Elution mode
    • B01D15/426Specific type of solvent
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • C07K1/18Ion-exchange chromatography
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065Purification, fragmentation
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/96Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/50Immunoglobulins specific features characterized by immunoglobulin fragments

Abstract

A method for the purification of immunoglobulins by ion exchange chromatography is described. The chromatographic method uses a weak ion exchange resin and a single step elution process for the purification of an immunoglobulin. Additionally a method for the determination of the salt concentration for the single step elution of an immunoglobulin from an ion exchange resin is described.

Description

    FIELD OF THE INVENTION
  • [0001]
    The current invention relates to the field of polypeptide purification. A novel method for the purification of immunoglobulins with an ion exchange resin is described herein. At the same time a method for the fast determination of purification conditions is given.
  • BACKGROUND OF THE INVENTION
  • [0002]
    Proteins and especially immunoglobulins play an important role in today's medical portfolio. For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans nucleic acids, viruses, and host cell proteins, which would cause severe harm, have to be removed especially. To meet the regulatory specification one or more purification steps have to follow the manufacturing process. Among other, purity, throughput, and yield play an important role in determining an appropriate purification process.
  • [0003]
    Different methods are well established and widespread used for protein purification, such as affinity chromatography with microbial proteins (e.g. protein A or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. with phenyl-sepharose, aza-arenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g. with Ni(II)- and Cu(II)-affinity material), size exclusion chromatography, and electrophoretical methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M. A., Appl. Biochem. Biotech. 75 (1998) 93-102).
  • [0004]
    Necina, R. et al., Biotechnol. Bioeng. 60 (1998) 689-698, reported the capture of human monoclonal antibodies directly from cell culture supernatants by ion exchange media exhibiting high charge density. In WO 89/05157 a method is reported for the purification of product immunoglobulins by directly subjecting the cell culture medium to a cation exchange treatment. A one-step purification of monoclonal IgG antibodies from mouse ascites is described by Danielsson, A., et al., J. Immun. Meth. 115 (1988), 79-88.
  • [0005]
    A combination of methods, i.e. a caprylic acid precipitation and ion exchange chromatography, was used by Raweerith, R. et al. (J. Immun. Meth. 282 (2003) 63-72), as means to fractionate pepsin-digested horse antivenom F(ab′)2 antibody. In WO 2003/102132 the combination of a non-affinity purification step and a high-performance tangential-flow filtration is reported for the purification of proteins. The combination of two affinity chromatography steps is reported in WO 92/19973.
  • [0006]
    Follman, D. K., and Fahrner, R. L., reported a factorial screening of antibody purification processes using three chromatography steps without protein A (J. Chrom. A 1024 (2004) 79-85). Mhatre, R. et al. (J. Chrom. A 707 (1995) 225-231), explored the purification of antibody Fab fragments by cation exchange chromatography and pH gradient elution.
  • [0007]
    WO 94/00561 reports human monoclonal anti-rhesus antibodies and cell lines producing the same. A method for purifying a polypeptide by ion exchange chromatography is reported in WO 2004/024866 in which a gradient wash is used to resolve a polypeptide of interest from one or more contaminants. Schwarz, A. et al. (Laborpraxis 21 (1997) 62-66), report the purification of monoclonal antibodies with a CM-HyperD-column.
  • [0008]
    WO 2004/076485 reports a process for antibody purification by protein A and ion exchange chromatography. In EP 0 530 447 a process for purifying IgG monoclonal antibodies by a combination of three chromatographic steps is reported. The removal of protein A from antibody preparations is reported in U.S. Pat. No. 4,983,722.
  • [0009]
    WO 95/16037 reports the purification of anti-EGF-R/anti-CD3 bispecific monoclonal antibodies from hybrid hybridoma performed by protein A cation exchange chromatography. The separation of antibody monomers from its multimers by use of ion exchange chromatography is reported in EP 1 084 136. U.S. Pat. No. 5,429,746 relates to the application of hydrophobic interaction chromatography combination chromatography to the purification of antibody molecule proteins.
  • SUMMARY OF THE INVENTION
  • [0010]
    Thus it is the objective of the current invention to provide another method for the purification of recombinantly produced immunoglobulins and for the separation of monomeric and multimeric immunoglobulin species.
  • [0011]
    The current invention provides a method for purifying an immunoglobulin, wherein the method comprises the following steps
      • a) providing a solution comprising an immunoglobulin, a buffer substance, and optionally a salt;
      • b) bringing the solution and a weak ion exchange material in contact under conditions whereby the immunoglobulin binds to the weak ion exchange material;
      • c) recovering the immunoglobulin from the weak ion exchange material in a single step by using a solution comprising a buffer substance and a salt.
  • [0015]
    The invention further provides a method for determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process, comprising the following two steps
      • a) step one comprising the following sub-steps
        • a1) providing a solution comprising a polypeptide, a buffer substance, and optionally a salt;
        • a2) bringing a first aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
        • a3) recovering the polypeptide from the ion exchange material by using a solution comprising a buffer substance and a salt whereby the concentration of the salt is increased linearly;
        • a4) determining the starting concentration of the salt where the first fraction of the polypeptide starts to elute from the ion exchange column;
      • b) step two comprising the following sub-steps
        • b1) bringing a second aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
        • b2) recovering the polypeptide from the ion exchange material by using a three step elution method, whereby
          • i) the salt concentration of the first elution step is calculated as the sum of
            • the product of the starting concentration of the salt as determined in step a4) and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt and
            • the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt;
          • ii) the salt concentration of the second elution step is the product of the salt concentration of the first elution step and a factor between 1.25 and 1.35;
          • iii) the salt concentration of the third elution step is the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70;
        • whereby the factors of step ii) and iii) are determined as follows: at a starting concentration between 10 mM and 40 mM the factors are 1.35 and 1.70 respectively, at a starting concentration between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at a starting concentration of more than 70 mM the factors are 1.25 and 1.50 respectively.
        • b3) determining at which sub-step of the three step elution method of step b2) the polypeptide is eluted from the ion exchange column thereby determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process.
    DETAILED DESCRIPTION OF THE INVENTION
  • [0031]
    These terms are used within this application in accordance with the following definition:
  • [0032]
    The term “ion exchange resin” or “ion exchange material” as used within this application denotes an immobile high molecular weight matrix that carries covalently bound charged substituents. For overall charge neutrality not covalently bound counter ions are bound thereto. The “ion exchange material” has the ability to exchange its not covalently bound counter ions for similarly charged ions of the surrounding solution. Depending on the charge of its exchangeable counter ions the “ion exchange resin” is referred to as cation exchange resin or as anion exchange resin. Depending on the nature of the charged group (substituent) the “ion exchange resin” is referred to as, e.g. in the case of cation exchange resins, sulfonic acid resin (S), or carboxymethyl resin (CM). Depending on the chemical nature of the charged group/substituent the “ion exchange resin” can additionally be classified as strong or weak ion exchange resin, depending on the strength of the covalently bound charged substituent. For example, strong cation exchange resins have a sulfonic acid group as charged substituent, weak cation exchange resins have a carboxylic group, preferably a carboxymethyl group, as charged substituent, and weak anion exchange resins have a diethylaminoethyl group as charged substituent.
  • [0033]
    Cation exchange resins are available under different names from a multitude of companies such as e.g. Bio-Rex, Macro-Prep CM (available from Biorad Laboratories, Hercules, Calif., USA), weak cation exchanger WCX 2 (available from Ciphergen, Fremont, Calif., USA), Dowex® MAC-3 (available from Dow chemical company—liquid separations, Midland, Mich., USA), Mustang C (available from Pall Corporation, East Hills, N.Y., USA), Cellulose CM-23, CM-32, CM-52, hyper-D, and partisphere (available from Whatman plc, Brentford, UK), Amberlite® IRC 76, IRC 747, IRC 748, GT 73 (available from Tosoh Bioscience GmbH, Stuttgart, Germany), CM 1500, CM 3000 (available from BioChrom Labs, Terre Haute, Ind., USA), and CM-Sepharose™ Fast Flow (available from GE Healthcare—Amersham Biosciences Europe GmbH, Freiburg, Germany).
  • [0034]
    Preferably, the charged substituents of the weak ion exchange material are at least about 90% carboxylic acid groups, more than 90% carboxylic acid groups, or more than 95% carboxylic acid groups.
  • [0035]
    The terms “single step elution” and “single step gradient elution”, which are used interchangeably within this application, denote a method wherein e.g. the concentration of a substance causing elution, i.e. the dissolution of a bound compound from a material, is raised or lowered at once, i.e. directly from a starting value/level to a final value/level, i.e. in a single step.
  • [0036]
    A “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 20 amino acid residues are referred to as “peptides.”
  • [0037]
    A “protein” is a macromolecule comprising one or more polypeptide chains or a polypeptide chain of more than 100 amino acid residues. A protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • [0038]
    The terms “antibody” and “immunoglobulin” which can be used interchangeably within this application comprise at least two light polypeptide chains and two heavy polypeptide chains. Each of the heavy and light polypeptide chains contains a variable region (generally the amino terminal portion of the polypeptide chain) which contains a binding domain for interaction with the antigen. Each of the heavy and light polypeptide chains also comprises a constant region (generally the carboxyl terminal portion) which may mediate the binding of the antibody to host tissues or factors including various cells of the immune system, some phagocytic cells and a first component (C1q) of the classical complement system. Typically, the light and heavy polypeptide chains are complete chains, each consisting essentially of a variable region, i.e. VL or VH, and a complete constant region, i.e. of CL in case of a light polypeptide chain or of CH 1, CH 2, CH 3, and optionally CH 4 in case of a heavy polypeptide chain. The variable regions of the antibody according to the invention can be grafted to constant regions of other isotypes. For example, a polynucleotide encoding the variable region of a heavy chain of the 1-isotype can be grafted to polynucleotide encoding the constant region of another heavy chain class (or subclass).
  • [0039]
    As used herein, the term “antibody” or “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by antibody genes. The recognized antibody genes include the different constant region genes as well as the myriad antibody variable region genes. Antibodies may exist in a variety of forms, including, for example, Fv, Fab, and F(ab)2 as well as single chains (e.g. Huston, J. S., et al., PNAS USA 85 (1988) 5879-5883; Bird et al., Science 242 (1988) 423-426; and, in general, Hood et al., Immunology, Benjamin N.Y., 2nd edition (1984) and Hunkapiller and Hood, Nature 323 (1986) 15-16). In one embodiment antibodies according to the invention comprise monoclonal antibodies and fragments thereof, for example isolated heavy or light chains, or heavy or light chains only consisting of constant regions as well as fragments thereof.
  • [0040]
    General chromatographic methods and their use are known to a person skilled in the art. See for example, Chromatography, 5th edition, Part A: Fundamentals and Techniques, Heftmann, E. (ed), Elsevier Science Publishing Company, New York, (1992); Advanced Chromatographic and Electromigration Methods in Biosciences, Deyl, Z. (ed.), Elsevier Science BV, Amsterdam, The Netherlands, (1998); Chromatography Today, Poole, C. F., and Poole, S. K., Elsevier Science Publishing Company, New York, (1991); Scopes, Protein Purification: Principles and Practice (1982); Sambrook, J., et al. (ed), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; or Current Protocols in Molecular Biology, Ausubel, F. M., et al. (eds), John Wiley & Sons, Inc., New York.
  • [0041]
    The current invention provides a method for purifying an immunoglobulin, wherein the method comprises the following steps
      • a) providing a solution comprising an immunoglobulin, a buffer substance, and optionally a salt;
      • b) bringing the solution and a weak ion exchange material in contact under conditions whereby the immunoglobulin binds to the weak ion exchange material;
      • c) recovering the immunoglobulin from the weak ion exchange material in a single step by using a solution comprising a buffer substance and a salt.
  • [0045]
    The purification process of immunoglobulins in general comprises a multistep chromatographic part. In the first step non-immunoglobulin polypeptides and proteins are separated from the immunoglobulin fraction by an affinity chromatography, e.g. with protein A. Afterwards an ion exchange chromatography can be performed to disunite the individual immunoglobulin classes and to remove traces of protein A, which has been coeluted from the first column. Finally a third chromatographic step is necessary to separate immunoglobulin monomers from multimers and fragments of the same class. Sometimes the amount of aggregates is high (5% or more) and it is not possible to separate them efficiently in the third purification step necessitating further purification steps.
  • [0046]
    With the recombinant production of specific immunoglobulins the separation step for the separation of different immunoglobulin classes is dispensable. Thus the overall purification process of recombinantly produced immunoglobulins may be reduced to two chromatographic steps.
  • [0047]
    The conditioned protein A eluate is in general chromatographically processed on a cation exchange material at pH values below the isoelectric point of the respective immunoglobulin protein.
  • [0048]
    The anti IL-1R antibody (WO 2005/023872) and Herceptin®, an anti-HER2 antibody (WO 99/57134), were available in sufficient quantities in our laboratories at the time of the invention and therefore the current invention is exemplified with these two immunoglobulins. Likewise the invention is in general practicable with immunoglobulins. This exemplified description is done only by way of example and not by way of limitation of the invention. These examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.
  • [0049]
    The current invention describes a purification method for the separation of immunoglobulin monomers from aggregates and fragments as well as the depletion of other polypeptide impurities. As can be seen from the experiments this purification is achieved by the use of weak ion exchange resins, preferably by the use of weak cation exchange resins. As exemplified by the comparison of strong and weak ion exchange materials the weak ion exchange material provides a separation of monomeric and aggregated forms of the immunoglobulins (see examples 1 and 2 and examples 9 and 12).
  • [0050]
    In one embodiment of the invention the pH value of the buffer material (substance) is of from pH 3.0 to pH 10.0, preferably of from pH 3.0 to pH 7.0, more preferred of from pH 4.0 to pH 6.0 and most preferred of from pH 4.5 to pH 5.5.
  • [0051]
    In one embodiment the pH value is kept constant in the single step, i.e. it is maintained at the same value in the single step.
  • [0052]
    Another preferred item of the current invention is that the method of the current invention is applicable to immunoglobulins that have an isoelectric point (pI) of 6.0 or more (pI≧6.0) and therefore have a net positive charge in the pH range starting from pH 6.0 to pH 14.
  • [0053]
    A preferred embodiment of the invention is the purification of an immunoglobulin of the IgG or IgE class.
  • [0054]
    A weak cation exchange material is used in a preferred embodiment of the current invention.
  • [0055]
    The buffer material is preferably employed in a concentration range between 5 mM and 100 mM as exemplified.
  • [0056]
    For the recovering of the bound immunoglobulins from the weak ion exchange material the conductivity of the buffer/solution is increased. This can be accomplished either by an increased buffer salt concentration or by the addition of other salts, so called elution salts, to the buffer solution. Preferred elution salts are sodium citrate, sodium chloride, sodium sulphate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate, as well as other salts of citric and phosphoric acid, and any mixture of these components. Especially preferred are sodium citrate, sodium chloride, potassium chloride and mixtures thereof.
  • [0057]
    The concentration of the salt, causing the elution, is preferably in the range of between 5 mM and 500 mM, more preferred between 5 mM and 400 mM, and especially preferred between 5 mM and 250 mM.
  • [0058]
    Another preferred embodiment of the invention is the use of the salt, causing the elution, at the same time as buffer substance, especially with citric acid and salts thereof or phosphoric acid and salts thereof.
  • [0059]
    The method of the current invention is preferably a chromatographic or batch method, especially preferred is a method comprising a batch elution.
  • [0060]
    Another preferred embodiment of the current invention is that the purification is a single step purification process.
  • [0061]
    A “single step” denotes a process wherein one or more conditions, for example the pH, the ionic strength, concentration of a salt, and/or the flow of a chromatography, is/are changed all at once from a starting value to a final value, i.e. the conditions are changed incrementally, i.e. stepwise, in contrast to a linear change.
  • [0062]
    Still a preferred embodiment of the current invention is that the method comprises the additional step of a purification of the immunoglobulin by a protein A affinity chromatography before step a) of the method.
  • [0063]
    The current invention further provides a method for determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process, comprising the following two steps
      • a) step one comprising the following sub-steps
        • a1) providing a solution comprising a polypeptide, a buffer substance, and optionally a salt;
        • a2) bringing a first aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
        • a3) recovering the polypeptide from the ion exchange material by using a solution comprising a buffer substance and a salt whereby the concentration of the salt is increased linearly;
        • a4) determining the starting concentration of the salt where the first fraction of the polypeptide starts to elute from the ion exchange column;
      • b) step two comprising the following sub-steps
        • b1) bringing a second aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
        • b2) recovering the polypeptide from the ion exchange material by using a three step elution method, whereby
          • i) the salt concentration of the first elution step is calculated as the sum of
            • the product of the starting concentration of the salt as determined in step a4) and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt and
            • the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt;
          • ii) the salt concentration of the second elution step is the product of the salt concentration of the first elution step and a factor between 1.25 and 1.35;
          • iii) the salt concentration of the third elution step is the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70;
        • whereby the factors of step ii) and iii) are determined as follows: at a starting concentration between 10 mM and 40 mM the factors are 1.35 and 1.70 respectively, at a starting concentration between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at a staring concentrations of more than 70 mM the factors are 1.25 and 1.50 respectively.
        • b3) determining at which sub-step of the three step elution method of step b2) the polypeptide is eluted from the ion exchange column thereby determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process.
  • [0079]
    The factors of step b2) define a range that has been determined experimentally. These values are no absolute values but merely a target value. A deviation of 10% is maintainable.
  • [0080]
    The current invention describes a method for determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process for the purification of polypeptides from other proteinaceous material.
  • [0081]
    The concentration, at which the elution of the polypeptide, preferably of an immunoglobulin, from the ion exchange resin starts, provides the basis for the second optimization step b), a three step elution method. The approximate buffer/salt concentrations for the step elution are calculated as follows:
      • the salt concentration of the first elution step is equal to the sum of
        • as first summand the product of the concentration of the salt, at which the elution from the ion exchange column starts as determined with the linear increasing salt gradient, and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt causing the elution and
        • as second summand the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt;
      • the salt concentration of the second elution step is equal to the product of the salt concentration of the first elution step and a factor of between 1.25 and 1.35;
      • the salt concentration of the third elution step is equal to the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70.
  • [0087]
    The factor included in the calculation of the concentration steps accounts for the interval between the concentration levels and is adjusted depending on the starting concentration. At small starting concentrations, i.e. between 10 mM and 40 mM, the factors are 1.35 and 1.70 respectively, at medium starting concentrations between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at high starting concentrations of more than 70 mM the factors are 1.25 and 1.50 respectively. These factors define a range that has been determined experimentally. These values are no absolute values but merely a target value. A deviation of 10% is maintainable.
  • [0088]
    The buffer salt has to be accounted for in the calculation because it is possible, as outlined in example 3, that the elution of a protein from an ion exchange resin can be effected by a change of the buffer salt concentration during the chromatography. If the buffer salt concentration is kept constant during the chromatography or is small compared to the stating concentration of the salt causing the elution it may be neglected during the calculation to reduce complexity.
  • [0089]
    In one embodiment the salt causing the elution is not the buffer salt and the salt concentration of step b2i) is the product of the concentration of the salt, at which the elution from the ion exchange column starts as determined with the linear increasing salt gradient in step a4), and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt causing the elution.
  • [0090]
    The calculation will be exemplified based on example 4 with the anti IL-1R antibody. With a starting concentration of 15 mM sodium citrate, as determined in example 3, consisting of a 10 mM buffer concentration and a 5 mM contribution from the linear gradient, the three steps are calculated as follows:
      • the target concentration for step one is calculated to be 30 mM (5 mM*2+10 mM*2) sodium citrate
        • in detail: 5 mM (starting concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula) plus 10 mM (buffer salt concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula)
      • the target concentration for step two is calculated to be 40.5 mM (30 mM*1.35) sodium citrate
        • in detail: 30 mM sodium citrate is the concentration of step one multiplied by 1.35 (the starting concentration is 15 mM, therefore as factor 1.35 is selected)
      • the target concentration for step three is calculated to be 51 mM (30 mM*1.70) sodium citrate
        • in detail: 30 mM sodium citrate is the concentration of step one multiplied by 1.70 (the starting concentration is 15 mM, therefore as factor 1.70 is selected)
  • [0097]
    As can be seen from the experiments this purification is achieved in a preferred embodiment by the use of a weak ion exchange material, especially preferred of a weak cation exchange material.
  • [0098]
    A preferred embodiment of the invention is that the polypeptide is an immunoglobulin, especially preferred an immunoglobulin of the IgG or IgE class.
  • [0099]
    In one embodiment of the invention the pH value of the buffer material/substance is of from pH 3.0 to pH 10.0, preferably of from pH 3.0 to pH 7.0, more preferred of from pH 4.0 to pH 6.0 and most preferred of from pH 4.5 to pH 5.5.
  • [0100]
    Another preferred item of the current invention is that the method of the current invention is applicable to immunoglobulins that have an isoelectric point (pI) of 6.0 or more (pI≧6.0) and therefore have a net positive charge in the pH range starting from pH 6.0 to pH 14.
  • [0101]
    The buffer material/substance is preferably employed in a concentration range between 5 mM and 100 mM as exemplified.
  • [0102]
    For the recovering of the bound immunoglobulins from the ion exchange material the conductivity of the buffer/solution is increased. This can be accomplished either by an increased buffer salt concentration or by the addition of other salts, so called elution salts, to the buffer solution. Preferred elution salts are sodium citrate, sodium chloride, sodium sulphate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate, as well as other salts of citric and phosphoric acid, and any mixture of these components. Especially preferred are sodium citrate, sodium chloride, potassium chloride and any mixture thereof.
  • [0103]
    The concentration of the salt, causing the elution, is preferably in the range of between 5 mM and 500 mM, more preferred between 5 mM and 400 mM, and especially preferred between 5 mM and 250 mM.
  • [0104]
    Another preferred embodiment of the invention is the use of the salt, causing the elution, at the same time as buffer substance, especially with citric acid and salts thereof or phosphoric acid and salts thereof.
  • [0105]
    The method of the current invention is preferably a chromatographic or batch method, especially preferred is a method comprising a batch elution.
  • [0106]
    Another preferred embodiment of the current invention is that the purification is a single step purification process.
  • [0107]
    Still a preferred embodiment of the current invention is that the method comprises the additional step of a purification of the immunoglobulin by a protein A affinity chromatography before step a) of the method.
  • [0108]
    The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.
  • DESCRIPTION OF THE FIGURES
  • [0109]
    FIG. 1 Single step elution of anti IL-1R antibody from strong cation exchange resin SP-Sepharose; monomeric and aggregated forms of the receptor antibody are not separated and elute as one peak.
  • [0110]
    FIG. 2 Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A.
  • [0111]
    FIG. 3 Linear gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl with sodium citrate at pH 5.0; monomeric and aggregated forms of the receptor antibody are partially separated and elute at a sodium citrate starting concentration of 15 mM as a main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising a mixture of monomeric form, aggregated forms of the immunoglobulin, and protein A. The SEC analysis of the two fractions is inserted into the ion exchange chromatogram. In the main peak aggregates are absent. In contrast in the second peak aggregated forms of the immunoglobulin are present.
  • [0112]
    FIG. 4 Three step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Toyopearl with sodium citrate at pH 5.0; monomeric and aggregated forms of the receptor antibody are partially separated and elute as a main peak comprising the monomeric form of the immunoglobulin at a sodium citrate concentration of 34 mM and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A at a sodium citrate concentration of 44 mM.
  • [0113]
    FIG. 5 Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 5.5; monomeric and aggregated forms of the receptor antibody are separated and elute as a main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as protein A.
  • [0114]
    FIG. 6 a Elution profile on a CM-Sepharose fast flow; two peaks can be identified: a main peak, corresponding to the monomeric anti IL-1R antibody, and a smaller second peak, that contains mainly aggregates and other impurities.
  • [0115]
    FIG. 6 b Elution profile of anti IL-1R antibody on a SP-Sepharose fast flow; one peak can be identified which contains monomeric immunoglobulin, aggregates and other impurities which have not been separated on this column.
  • [0116]
    FIG. 7 a Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 100 mM sodium chloride at pH 5.5.
  • [0117]
    FIG. 7 b Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 5.5.
  • [0118]
    FIG. 7 c Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 200 mM sodium chloride at pH 5.5.
  • [0119]
    FIG. 8 a Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 4.0.
  • [0120]
    FIG. 8 b Single step gradient elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose with 150 mM sodium chloride at pH 6.0.
  • [0121]
    FIG. 9 a Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; size exclusion chromatography (SEC) of the starting material showing both monomeric and aggregated forms of the immunoglobulin.
  • [0122]
    FIG. 9 b Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as other protein. In this figure the size exclusion chromatography of the first (main) peak is shown. Only one peak is eluted from the SEC-column, which is the monomeric immunoglobulin.
  • [0123]
    FIG. 9 c Single step elution of anti IL-1R antibody from weak cation exchange resin CM-Sepharose; monomeric and aggregated forms of the receptor antibody are partially separated and elute as one main peak comprising the monomeric form of the immunoglobulin and as a second peak, comprising monomeric and aggregated forms of the immunoglobulin as well as other protein. In this figure the size exclusion chromatography of the second peak is shown. In the chromatogram at least three peaks can be seen, equivalent to monomeric and aggregated forms of the immunoglobulin and other protein.
  • [0124]
    FIG. 10 Single step elution of anti-HER-2 antibody from strong cation exchange resin SP-Sepharose; monomeric and aggregated forms of the antibody are not separated and elute as one peak.
  • [0125]
    FIG. 11 Linear gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with sodium chloride in sodium citrate buffer at pH 5.5; monomeric and aggregated forms of the antibody are partially separated and elute with a starting concentration of 80 mM sodium chloride as one main peak comprising the monomeric form of the immunoglobulin; the monomeric and aggregated forms elute as mixture in the tailing of the main peak together with protein A.
  • [0126]
    FIG. 12 Three step gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with sodium chloride in sodium citrate buffer at pH 5.5; monomeric and aggregated forms of the antibody are separated and elute as one main peak comprising the monomeric form of the immunoglobulin at a sodium chloride concentration of 80 mM and as a second peak comprising monomeric and aggregated forms of the immunoglobulin as well as protein A at a sodium chloride concentration of 100 mM.
  • [0127]
    FIG. 13 Single step gradient elution of anti-HER-2 antibody from weak cation exchange resin CM-Sepharose with 80 mM sodium chloride at pH 5.5; the monomeric form is eluted free of aggregated forms; the aggregated forms elute after a second sodium chloride step to 120 mM as a second defined peak.
  • [0128]
    FIG. 14 Single step gradient elution of anti-HER-2 antibody from strong cation exchange resin SP-Sepharose with 80 mM sodium chloride at pH 5.5; two peaks are obtained: peak 1 contains only monomeric Herceptin®, peak 2, which is eluted after a second sodium chloride step to 120 mM, contains a high amount of monomeric Herceptin® and aggregates; the yield of Herceptin® in its monomeric form is less than 60%.
  • EXPERIMENTAL PART Material
  • [0129]
    An IgG4 immunoglobulin anti IL-1R antibody (hereinafter referred to as immunoglobulin, WO 2005/023872) was purified in a first step with a protein A affinity chromatography. Elution from the protein A column was carried out under acidic conditions (10 mM sodium citrate buffer, pH value 3.0±0.5). Before the filtration step the pH value of the fraction containing the immunoglobulin was adjusted with a concentrated, e.g. 1 M, buffer solution of pH 9.0 (e.g. tris-hydroxymethyl-aminomethane (TRIS) or phosphate buffer) to pH 5.0. The protein A eluate is a solution with a protein concentration between 5 mg/ml and 15 mg/ml and is buffered with sodium citrate. This material is referred to in the following as conditioned protein A eluate, which is prepared for loading onto a cationic exchange resin.
  • Example 1
  • [0130]
    In this comparative example an ion exchange chromatography with a strong cation exchange resin and single step elution is described.
  • Chromatographic Conditions:
  • [0131]
  • [0000]
    Resin: SP-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Loading: max. 20 g protein/L gel matrix
    Wash step: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Elution: 25 mM sodium citrate buffer with 100 mM sodium
    chloride, adjusted to pH 5.0
  • [0132]
    The conditioned protein A eluate was applied to a chromatography column containing a strong cation exchange resin (SP-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value was kept constant and the conductivity was varied (increased) by the addition of sodium chloride.
  • [0133]
    In FIG. 1 the elution chromatogram of the cation exchange chromatography of the anti IL-1R antibody on the strong cation exchange resin SP-Sepharose is presented. The elution is a single step replacement elution with sodium chloride without altering the pH value of the chromatographic system. The monomeric and aggregated immunoglobulin molecules are not separated and thus with this method no purification by reduction of the aggregate content in the eluate compared to the loaded material can be obtained.
  • Example 2
  • [0134]
    In this example an ion exchange chromatography with a weak cation exchange resin and single step elution is described.
  • [0135]
    To achieve a separation of monomeric and aggregated forms of an immunoglobulin, a weak cation exchange resin was employed. By using this kind of resin an increase of the conductivity by a step elution is accompanied by a particular pH shift on the resin (even when the pH of the eluting buffer remains constant). This effect facilitates the discrimination e.g. between monomeric immunoglobulins and aggregated forms. Furthermore, other impurities, like traces of host cell proteins or protein A, can efficiently be separated from the monomeric mean fraction, without significant loss in yield.
  • Chromatographic Conditions:
  • [0136]
  • [0000]
    Resin: CM-Toyopearl
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Elution: 25 mM sodium citrate buffer with 100 mM sodium
    chloride, adjusted to pH 5.0
  • [0137]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value in the mobile phase was kept constant and the conductivity was varied by the addition of sodium chloride.
  • [0138]
    In FIG. 2 the elution profile with the same chromatographic conditions as in example 1 but this time with a weak cation exchange resin, CM-Toyopearl, is presented. Herein a second peak as shoulder of the main peak appears. This separation behavior is different to that with a strong cation exchange resin, such as SP-Sepharose. An analysis of fractions corresponding to the main peak and to the second shoulder peak showed a significant amount of aggregates to be present in the shoulder peak fraction. No aggregates were detectable in the main peak fractions (see also FIGS. 9 a to c).
  • Example 3 Optimization of the Chromatographic Method First Step: Linear Concentration Gradient
  • [0139]
    To optimize the cation exchange chromatography on a weak cation exchange material an optimization procedure, which is consisting of three steps, was put into practice:
  • [0140]
    The first step is a chromatography using a linear concentration gradient of the buffer salt, sodium citrate. Just as well is it possible to keep the concentration of the buffer salt constant and to admix a linearly increasing concentration of a salt causing the elution of the immunoglobulin. In both cases is the conductivity of the solution increased without an alteration of the pH value of the mobile phase. Salts suitable for the elution are e.g. sodium chloride, sodium sulphate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate, citric acid and salts thereof as well as mixtures of these components. The concentrations of from 10 mM to 500 mM, which are applied, are adjusted accordingly to set conductivity in the range of from about 1 milli S/cm to about 50 milli S/cm.
  • Chromatographic Conditions:
  • [0141]
  • [0000]
    Resin CM-Toyopearl
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Elution: linear gradient; from 10 mM sodium citrate buffer,
    adjusted to pH 5.0, to 100 mM sodium citrate buffer,
    adjusted to pH 5.0
  • [0142]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a linear gradient elution method, whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was raised linearly from 10 mM to 100 mM over 40 column volumes. After the final sodium citrate concentration was reached the elution was continued for an additional 40 column volumes.
  • [0143]
    In FIG. 3 the chromatogram of the linear buffer gradient elution of the anti IL-1R antibody is presented. The monomeric and the aggregated form of the immunoglobulin elute in a semi-detached peak starting at a concentration of 15 mM sodium citrate and ending at a concentration of 55 mM sodium citrate.
  • [0144]
    The recovery of the bound immunoglobulin from the cation exchange resin is depending on the conductivity of the applied solution. Therefore the cations of the buffer salt present in the eluting solution during the recovery step have to be considered as effecting the elution of the immunoglobulin from the cation exchange resin. The conductivity as well as the ionic strength of the mobile phase are effected by the total number of ions in the solution. Thus the number of monovalent cations different from hydrogen in one molecular formula of the employed buffer salt and the employed salt causing the elution have to be considered hereby.
  • Example 4 Optimization of the Chromatographic Method Second Step: Three Step Concentration Gradient Elution
  • [0145]
    The concentration, at which the elution of the immunoglobulin from the ion exchange resin starts, as determined in example 3, provides the basis for the second optimization step, a three step elution method. The approximate buffer/salt concentrations for the step elution are calculated as follows:
      • the salt concentration of the first elution step is equal to the sum of
        • as first summand the product of the concentration of the salt, at which the elution from the ion exchange column starts as determined with the linear increasing salt gradient, and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt causing the elution and
        • as second summand the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt;
      • the salt concentration of the second elution step is equal to the product of the salt concentration of the first elution step and a factor of between 1.25 and 1.35;
      • the salt concentration of the third elution step is equal to the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70.
  • [0151]
    The factor included in the calculation of the concentration steps accounts for the interval between the concentration levels and is adjusted depending on the starting concentration. At small starting concentrations, i.e. between 10 mM and 40 mM, the factors are 1.35 and 1.70 respectively, at medium starting concentrations between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at high starting concentrations of more than 70 mM the factors are 1.25 and 1.50 respectively.
  • [0152]
    The factors define a range that has been determined experimentally. These values are no absolute values but merely a target value. A deviation of 10% is maintainable.
  • [0153]
    The buffer salt has to be accounted for in the calculation because it is possible as outlined in example 3 that the elution of a protein from an ion exchange resin can be effected by a change of the buffer salt concentration during the chromatography. If the buffer salt concentration is kept constant during the chromatography or is small compared to the stating concentration (≦15% of the salt concentration) it may be neglected during the calculation to reduce complexity.
  • [0154]
    With a starting concentration of 15 mM sodium citrate, as determined in example 3, consisting of a 10 mM buffer concentration and a 5 mM contribution from the linear gradient, the three steps can be calculated as follows:
      • the target concentration for step 1 is calculated to be 30 mM (5 mM*2+10 mM*2) sodium citrate
        • in detail: 5 mM (starting concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula) plus 10 mM (buffer salt concentration) multiplied with two (citric acid is a trivalent acid, employed as di-sodium salt; therefore two monovalent cations different from hydrogen are present in the molecular formula)
      • the target concentration for step 2 is calculated to be 40.5 mM (30 mM*1.35) sodium citrate
        • in detail: 30 mM sodium citrate is the concentration of step 1 multiplied by 1.35 (the starting concentration is 15 mM, therefore as factor 1.35 is selected)
      • the target concentration for step 3 is calculated to be 51 mM (30 mM*1.70) sodium citrate
        • in detail: 30 mM sodium citrate is the concentration of step 1 multiplied by 1.70 (the starting concentration is 15 mM, therefore as factor 1.70 is selected)
    Chromatographic Conditions:
  • [0161]
  • [0000]
    Resin: CM-Toyopearl
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.0
    Elution: step 1: 34 mM sodium citrate buffer, adjusted to pH 5.0
    step 2: 44 mM sodium citrate buffer, adjusted to pH 5.0
    step 3: 54 mM sodium citrate buffer, adjusted to pH 5.0
  • [0162]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Toyopearl). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a step gradient elution method (=a method wherein the concentration of the elution salt is changed stepwise from a starting value/level to a final value/level), whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was raised from 10 mM as starting condition to 34 mM in the first step, to 44 mM in the second step, and to 54 mM in the final step. After each increase of the salt concentration ten column volumes of the elution buffer were passed through the column prior to the next step. After the final sodium citrate concentration was reached the elution was continued for an additional 10 column volumes.
  • [0163]
    In FIG. 4 the elution profile of the three step gradient elution of anti IL-1R antibody is presented. The monomeric immunoglobulin elutes in the first step fraction and the aggregates elute in the second step fraction.
  • Example 5 Optimization of the Chromatographic Method Third Step: Single Step Elution with Sodium Chloride
  • [0164]
    The final step of the optimization procedure is the adaptation to a single step elution method (=a method wherein the concentration of the elution salt is changed at once from a starting value to a final value). For this purpose the pH of the chromatography is raised from 5.0 to 5.5. This pH shift improves the separation from protein A, due that protein A has an isoelectric point below 5.5. Additionally the elution salt is changed from sodium citrate, which is further on used as buffer salt, to sodium chloride. Additional analyses have been carried out (DNA, host cell protein, protein A content, and glycosylation pattern with LC-MS) with the fractions after this chromatographic run.
  • Chromatographic Conditions:
  • [0165]
  • [0000]
    Resin: CM-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate, adjusted to pH 5.5
    Elution: 10 mM sodium citrate with 150 mM sodium
    chloride, adjusted to pH 5.5
  • [0166]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method (=a method wherein the concentration of the elution salt is changed at once from a starting value to a final value), whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and 150 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin.
  • [0167]
    The elution chromatogram of the single step elution with sodium chloride is presented in FIG. 5. The single step gradient chromatography effects resolution of the main monomeric fraction and the aggregate/protein A fraction. The yield of monomeric immunoglobulin is more than 80%. Even more than 95% yield is possible.
  • Example 6 Comparison Between the Separation with a Strong Cation Exchange Resin (SP-Sepharose Fast Flow) and a Weak Cation Exchange Resin (CM-Sepharose Fast Flow)
  • [0168]
    A comparison between the strong SP-Sepharose ff exchanger and CM-Sepharose ff was done. Experiments were performed according to example 5 in duplicates (only one from each column is shown in FIGS. 6 a and b) and additional analyses have been carried out (DNA, host cell protein, protein A content, and glycosylation pattern with LC-MS).
  • Analytical Methods:
  • [0169]
  • [0000]
    Size Exclusion resin: TSK 3000 (Tosohaas)
    Chromatography: column: 300 × 7.8 mm
    flow rate: 0.5 ml/min
    buffer: 200 mM potassium phosphate
    containing
    250 mM potassium chloride,
    adjusted to pH 7.0
    • DNA-threshold-system: see e.g. Merrick, H., and Hawlitschek, G., Biotech Forum Europe 9 (1992) 398-403
    • Protein A ELISA: The wells of a micro titer plate are coated with a polyclonal protein A-IgG derived from chicken. After binding non-reacted antibody is removed by washing with sample buffer. For protein A binding a defined sample volume is added to the wells. The protein A present in the sample is bound by the chicken anti-body and retained in the wells of the plate. After the incubation the sample solution is removed and the wells are washed. For detection are added subsequently a chicken derived polyclonal anti-protein A-IgG-biotin conjugate and a streptavidin peroxidase conjugate. After a further washing step substrate solution is added resulting in the formation of a colored reaction product. The intensity of the color is proportional to the protein A content of the sample. After a defined time the reaction is stopped and the absorbance is measured.
    • Host cell protein (HCP) ELISA:
      • The walls of the wells of a micro titer plate are coated with a mixture of serum albumin and streptavidin. A goat derived polyclonal antibody against HCP is bound to the walls of the wells of the micro titer plate. After a washing step different wells of the micro titer plate are incubated with a HCP calibration sequence of different concentrations and sample solution. After the incubation not bound sample material is removed by washing with buffer solution. For the detection the wells are incubated with an antibody peroxidase conjugate to detect bound host cell protein. The fixed peroxidase activity is detected by incubation with ABTS and detection at 405 nm.
    Chromatographic Conditions:
  • [0174]
  • [0000]
    Resin: CM-Sepharose; SP-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Elution: 10 mM sodium citrate buffer with 150 mM sodium
    chloride, adjusted to pH 5.5
  • [0175]
    In FIGS. 6 a and 6 b a comparison between the elution chromatogram of a weak and a strong cation exchange resin is presented. Using a weak cation exchange resin (FIG. 6 a) a separation of the monomeric anti IL-1R antibody from other impurities is achieved. With the strong cation exchange resin (FIG. 6 b) no separation is possible under the same conditions. The fractions corresponding to the peaks have been collected and analyzed. The analysis results, which are listed in table 1, show that with the weak cation exchange resin aggregates and other impurities can effectively be depleted from the immunoglobulin preparation.
  • [0176]
    The data presented in table 1 show that it is possible to separate with a weak cation exchange resin monomeric anti IL-1R antibody from aggregated forms of the antibody. Furthermore DNA- and protein A-impurities can be depleted.
  • [0000]
    TABLE 1
    Analysis of the eluates: comparison between SP-Sepharose and CM-
    Sepharose, results of two different separations are presented.
    conditioned
    protein A SP-Sepharose eluate CM-Sepharose eluate
    analyte eluate single peak Peak 1 Peak 2
    amount of between 20 31 ng/mg 26 ng/mg 7.5 ng/mg 11 ng/mg 1638 ng/mg 550 ng/mg
    protein A and 50 ng/mg
    HCP between 20 3.88 ng/mg 3.98 ng/mg 3.13 ng/mg 3.27 ng/mg 946 ng/mg 1424 ng/mg
    and 120 ng/mg
    DNA between 36 pg/mg 16 pg/mg 157 pg/mg 131 pg/mg 1918 pg/mg 1222 pg/mg
    2800 and
    3500 pg/mg
    aggregates present present present not not present present
    present present in high in high
    amount amount
    mass no differences were found between SP- and CM-Sepharose
    analysis
  • Example 7 Comparative Example Elution at Different Conductivities Chromatographic Conditions:
  • [0177]
  • [0000]
    Resin: CM-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Elution: 10 mM sodium citrate buffer with 100 mM, 150 mM
    or 200 mM sodium chloride, adjusted to pH 5.5
  • [0178]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and in three different runs 100 mM, 150 mM, and 200 mM sodium chloride respectively were admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin. The elution chromatograms are displayed in FIGS. 7 a to c.
  • [0179]
    Good separations have been obtained using 150 mM sodium chloride and 200 mM sodium chloride as elution salt concentration.
  • Example 8 Comparative Example Elution at Different pH Values Chromatographic Conditions:
  • [0180]
  • [0000]
    Resin: GM-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Elution: 10 mM sodium citrate buffer with 150 mM sodium
    chloride, adjusted to pH 4.0, or 6.0
  • [0181]
    The conditioned protein A eluate was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase was kept constant at pH 4.0 or 6.0 respectively. The concentration of the buffer salt, sodium citrate, was kept constant, and 150 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer were passed through the column to elute the bound immunoglobulin. The elution chromatograms are displayed in FIGS. 8 a and b.
  • [0182]
    At pH 4.0 is the tendency to form aggregates of this immunoglobulin increased. But the CM-Sepharose is able to separate this higher amount of aggregates in two peaks.
  • Example 9 Chromatographic Separation of a Monoclonal Anti-HER-2 Antibody (WO 99/57134) with a Strong Cation Exchange Resin (SP-Sepharose)
  • [0183]
    The current invention is further exemplified in the following with Herceptin®, a monoclonal anti-HER-2 antibody.
  • [0184]
    The purification of Herceptin® with a cation exchange chromatography on SP-Sepharose, a strong cation exchange resin, was carried out. Under standard conditions of the current invention, i.e. step elution with e.g. sodium chloride, a separation of monomeric and aggregated forms of the antibody is not effected (FIG. 10).
  • Chromatographic Conditions:
  • [0185]
  • [0000]
    Resin: SP-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 25 mM 2-morpholinoethanesulfonic acid, 50 mM
    sodium chloride, adjusted to pH 5.6
    Loading: max. 20 g protein/L gel matrix
    Wash: 25 mM 2-morpholinoethanesulfonic acid, 50 mM
    sodium chloride, adjusted to pH 5.6
    Elution: 25 mM 2-morpholinoethanesulfonic acid, 95 mM
    sodium chloride, adjusted to pH 5.6
  • [0186]
    The monoclonal anti-HER-2 antibody (hereinafter referred to as Herceptin®) was purified in a first step with a protein A affinity chromatography. Elution from the protein A column is carried out under acidic conditions (10 mM sodium citrate buffer, pH value of 3.0±0.5). Before the filtration step the pH value of the fraction containing the antibody is adjusted with a concentrated tris-hydroxymethyl-aminomethane (TRIS) buffer to pH 5.6. The protein A eluate is a solution with a protein concentration between 5 mg/ml and 15 mg/ml and is buffered with sodium citrate.
  • [0187]
    The conditioned protein A eluate was applied to a chromatography column containing a strong cation exchange resin (SP-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step elution method, whereby the pH value was kept constant and the conductivity was varied by the (stepwise) increase of the sodium chloride concentration. The elution chromatogram is displayed in FIG. 10.
  • [0188]
    No separation of monomeric and aggregated forms of the antibody was achieved.
  • Example 10 Optimization of the Chromatographic Method First Step: Linear Concentration Gradient
  • [0189]
    To improve the separation of the two fractions the separation conditions have been optimized in accordance with the procedure as outlined with the anti IL-1R antibody.
  • [0190]
    In contrast to the anti IL-1R antibody optimization process a linear gradient of a (elution) salt, i.e. of sodium chloride, was used instead of a gradient of the buffer substance. The chromatogram of the linear sodium chloride gradient elution, which corresponds to the first step of the optimization procedure, is presented in FIG. 11. Analysis confirmed that the tail of the main peak is enriched with aggregated forms of the antibody.
  • Chromatographic Conditions:
  • [0191]
  • [0000]
    Resin CM-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Elution: linear gradient; from 10 mM sodium citrate buffer,
    adjusted to pH 5.5, to 10 mM sodium citrate buffer
    containing 400 mM sodium chloride, adjusted to pH 5.5
  • [0192]
    The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a linear gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt was kept constant. The concentration of the elution salt, sodium chloride, was raised linearly from 0 mM to 400 mM over 40 column volumes. The elution chromatogram is displayed in FIG. 11.
  • [0193]
    The replacement of the strong cation exchange resin by a weak cation exchange resin caused the detachment of a second peak as shoulder of the first main peak. This observation is similar to the observation in case of the anti IL-1R antibody.
  • [0194]
    The immunoglobulins start to elute from the column at a sodium chloride concentration of 80 mM.
  • Example 11 Optimization of the Chromatographic Method Second Step: Three Step Concentration Gradient Elution
  • [0195]
    The starting concentration, at which the immunoglobulin starts to elute, as determined in example 10 and as derived from the chromatogram presented in FIG. 11, is 80 mM sodium chloride. For the calculation of the three concentration steps for the second optimization step the buffer concentration can be neglected as it is low and kept constant during the chromatography.
  • [0196]
    The starting concentration of the sodium chloride is 80 mM and sodium chloride has one cation different from hydrogen in its molecular formula. Accordingly the concentrations for the three step elution are calculated to be 80 mM, 100 mM (=80 mM multiplied with 1.25), and 120 mM (=80 mM multiplied with 1.50) sodium chloride respectively.
  • Chromatographic Conditions:
  • [0197]
  • [0000]
    Resin: CM-Sepharose
    Flow rate: 160 cm/h
    Equilibration: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash: 10 mM sodium citrate buffer, adjusted to pH 5.5
    Elution: step 1: 10 mM sodium citrate buffer with 80 mM
    sodium chloride, adjusted to pH 5.5
    step 2: 10 mM sodium citrate buffer with 100 mM
    sodium chloride, adjusted to pH 5.5
    step 3: 10 mM sodium citrate buffer with 120 mM
    sodium chloride, adjusted to pH 5.5
  • [0198]
    The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a step gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt, sodium citrate, was kept constant. The concentration of the elution salt, sodium chloride, was raised from 0 mM as starting condition to 80 mM in the first step, to 100 mM in the second step, and to 120 mM in the final step. After each increase of the salt concentration ten column volumes of the elution buffer with the specified sodium chloride concentrations were passed through the column prior to the next concentration step. After the final sodium citrate concentration was reached the elution was continued for an additional 10 column volumes. The elution chromatogram is displayed in FIG. 12.
  • [0199]
    In the three step elution method the monomeric antibody is eluted at the step with a sodium chloride concentration of 80 mM. Size exclusion analysis confirmed that only monomeric antibody is eluted. After the sodium chloride concentration was increased to 100 mM in the second step, the aggregated forms eluted (FIG. 12).
  • Example 12 Optimization of the Chromatographic Method Third Step: Single Step Elution with Sodium Chloride Chromatographic Conditions:
  • [0200]
  • [0000]
    Chromatographic conditions:
    Resin: CM-Sepharose; SP-Sepharose
    Flow rate: 160 cm/h
    Equilibration:  10 mM sodium citrate, adjusted to pH 5.5
    Loading: max. 20 g protein/L gel matrix
    Wash:  10 mM sodium citrate, adjusted to pH 5.5
    Elution:  10 mM sodium citrate with 80 mM sodium chloride,
    adjusted to pH 5.5
  • [0201]
    The conditioned protein A eluate as described in example 9 was applied to a chromatography column containing a weak cation exchange resin (CM-Sepharose). After the loading step at a flow rate of 160 cm/h the column was washed with equilibration buffer (10 column volumes). The bound immunoglobulins were eluted with a single step gradient elution method, whereby the pH value in the mobile phase and the concentration of the buffer salt was kept constant. The concentration of the buffer salt, sodium citrate, was kept constant and 80 mM sodium chloride was admixed. After the increase of the salt concentration fifteen column volumes of the elution buffer with sodium chloride were passed through the column to elute the bound anti-HER-2 antibody in monomeric form. To affirm the separation of monomeric and aggregated forms of the antibody a second step, which is not necessary for the preparation of monomeric antibodies, to a sodium chloride concentration of 120 mM was performed. After this second increase the aggregated forms of the antibody eluted from the column. The elution chromatogram is displayed in FIG. 13.
  • [0202]
    If the same method is performed with a strong cation-exchange resin a significant loss of monomeric antibody is observed (yield of approximately 60% only compared to 95% and more on a weak cation exchange column), although the separation of monomeric and aggregated form of the antibody can be seen (FIG. 14).
  • [0203]
    Applying the conditions suitable for the separation on a weak cation exchange material to a strong cation exchange resin, SP-Sepharose, is not beneficial. Albeit the two fractions can be separated the yield of the monomeric antibody is reduced to 60% or even less.
  • [0000]
    TABLE 2
    Analysis of the eluates: comparison between SP-Sepharose and CM-
    Sepharose, results of two different separations are presented.
    conditioned
    protein A SP-Sepharose CM-Sepharose
    analyte eluate Peak 1 Peak 2 Peak 1 Peak 2
    amount of 17 ng/mg <3.9 ng/mg 5.7 ng/mg <3.9 ng/mg <3.9 ng/mg 32.2 ng/mg 39.7 ng/mg
    protein A
    HCP 3243 ng/mg 49.0 ng/mg 183.4 ng/mg 107.5 ng/mg 126.2 ng/mg 1247 ng/mg 988 ng/mg
    DNA 1615 pg/mg 830 pg/mg 2635 pg/mg 319 pg/mg 682 pg/mg 10554 pg/mg <9300 pg/mg
    aggregates 0.97% 0% 32% 0% 0% 15.2% 23.0%
    (SEC)

Claims (10)

  1. 1. Method for purifying an immunoglobulin, wherein the method comprises
    a) providing a solution comprising an immunoglobulin, a buffer substance, and optionally a salt;
    b) bringing the solution and a weak ion exchange material in contact under conditions whereby the immunoglobulin binds to the weak ion exchange material;
    c) recovering the immunoglobulin from the weak ion exchange material in a single step by using a solution comprising a buffer substance and a salt.
  2. 2. Method according to claim 1, characterized in that the weak ion exchange material is a weak cation exchange material.
  3. 3. Method according to any one of the claims 1 to 2, characterized in that the buffer substance is citric acid or a salt thereof or phosphoric acid or a salt thereof.
  4. 4. Method according to any one of the claims 1 to 3, characterized in that the method is a chromatographic or a batch method.
  5. 5. Method according to any one of the claims 1 to 4, characterized in that the salt in step c) is selected from the group consisting of sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, salts of citric acid, salts of phosphoric acid, and mixtures of these components.
  6. 6. Method according to any one of the claims 1 to 5, characterized in that the immunoglobulin is a member of the immunoglobulin class G or E.
  7. 7. Method according to any one of the claims 1 to 6, characterized in that the solution in the recovering step c) has a pH value of from pH 3.0 to pH 7.0.
  8. 8. Method for determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process, comprising the following two steps
    a) step one comprising the following sub-steps
    a1) providing a solution comprising a polypeptide, a buffer substance, and optionally a salt;
    a2) bringing a first aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
    a3) recovering the polypeptide from the ion exchange material by using a solution comprising a buffer substance and a salt whereby the concentration of the salt is increased linearly;
    a4) determining the starting concentration of the salt where the first fraction of the polypeptide starts to elute from the ion exchange column;
    b) step two comprising the following sub-steps
    b1) bringing a second aliquot of the solution containing the polypeptide and an ion exchange material in contact under conditions whereby the polypeptide binds to the ion exchange material;
    b2) recovering the polypeptide from the ion exchange material by using a three step elution method, whereby
    i) the salt concentration of the first elution step is calculated as the sum of
    the product of the starting concentration of the salt as determined in step a4) and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the salt and
    the product of the concentration of the buffer salt and the total number of monovalent cations different from hydrogen denoted in the molecular formula of the buffer salt;
    ii) the salt concentration of the second elution step is the product of the salt concentration of the first elution step and a factor between 1.25 and 1.35;
    iii) the salt concentration of the third elution step is the product of the salt concentration of the first elution step and a factor between 1.50 and 1.70;
    whereby the factors of step ii) and iii) are determined as follows: at a starting concentration of between 10 mM and 40 mM the factors are 1.35 and 1.70 respectively, at a starting concentration of between 40 mM and 70 mM the factors are 1.30 and 1.60 respectively, and at a starting concentration of more than 70 mM the factors are 1.25 and 1.50 respectively.
    b3) determining at which sub-step of the three step elution method of step b2) the polypeptide is eluted from the ion exchange column thereby determining the concentration of a salt for eluting a polypeptide from an ion exchange chromatography material in a single step purification process.
  9. 9. Method according to claim 8, characterized in that the ion exchange chromatography material is a weak ion exchange chromatography material.
  10. 10. Method according to any one of the claims 8 to 9, characterized in that the polypeptide is an immunoglobulin.
US11920288 2005-05-25 2006-05-23 Method for the Purification of Antibodies Abandoned US20090098660A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP05011302.6 2005-05-25
EP05011302 2005-05-25
PCT/EP2006/004863 WO2006125599A3 (en) 2005-05-25 2006-05-23 Method for the purification of antibodies

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2006/004863 A-371-Of-International WO2006125599A3 (en) 2005-05-25 2006-05-23 Method for the purification of antibodies

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14271058 Continuation US9631007B2 (en) 2005-05-25 2014-05-06 Method for the purification of antibodies

Publications (1)

Publication Number Publication Date
US20090098660A1 true true US20090098660A1 (en) 2009-04-16

Family

ID=34936911

Family Applications (3)

Application Number Title Priority Date Filing Date
US11920288 Abandoned US20090098660A1 (en) 2005-05-25 2006-05-23 Method for the Purification of Antibodies
US14271058 Active US9631007B2 (en) 2005-05-25 2014-05-06 Method for the purification of antibodies
US15458368 Pending US20170183394A1 (en) 2005-05-25 2017-03-14 Method for the purification of antibodies

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14271058 Active US9631007B2 (en) 2005-05-25 2014-05-06 Method for the purification of antibodies
US15458368 Pending US20170183394A1 (en) 2005-05-25 2017-03-14 Method for the purification of antibodies

Country Status (9)

Country Link
US (3) US20090098660A1 (en)
EP (2) EP1888636B1 (en)
JP (1) JP4852602B2 (en)
KR (1) KR100967778B1 (en)
CN (1) CN101180317B (en)
CA (1) CA2607375C (en)
DK (1) DK1888636T3 (en)
ES (2) ES2373402T3 (en)
WO (1) WO2006125599A3 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090118476A1 (en) * 2007-07-17 2009-05-07 Josef Burg Purification of pegylated polypeptides
US20110065903A1 (en) * 2007-07-17 2011-03-17 Josef Burg Chromatographic methods
CN103379949A (en) * 2010-10-11 2013-10-30 Abbvie 公司 Processes for purification of proteins

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101180317B (en) 2005-05-25 2013-06-19 弗·哈夫曼-拉罗切有限公司 Method for the purification of antibodies
EP2152745B1 (en) 2007-06-01 2014-01-08 F. Hoffmann-La Roche AG Immunoglobulin purification
CA2733782A1 (en) * 2008-08-14 2010-02-18 Merck Sharp & Dohme Corp. Methods for purifying antibodies using protein a affinity chromatography
ES2648264T3 (en) 2010-03-10 2017-12-29 F. Hoffmann-La Roche Ag Process for the purification of immunoglobulin solutions
RU2012146448A (en) * 2010-04-14 2014-05-20 Ф.Хоффманн-Ля Рош Аг The removal of aggregates of immunoglobulins
WO2012084829A1 (en) 2010-12-21 2012-06-28 F. Hoffmann-La Roche Ag Isoform enriched antibody preparation and method for obtaining it
WO2012123488A1 (en) 2011-03-16 2012-09-20 F. Hoffmann-La Roche Ag Ion exchange chromatography with improved selectivity for the separation of polypeptide monomers, aggregates and fragments by modulation of the mobile phase
EP2726496B1 (en) * 2011-07-01 2018-04-04 F.Hoffmann-La Roche Ag Method for separation of monomeric polypeptides from aggregated polypeptides
WO2015041218A1 (en) * 2013-09-17 2015-03-26 株式会社カネカ Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom
WO2015064971A1 (en) * 2013-10-30 2015-05-07 (주)셀트리온 Method for isolating isoforms of antibody using positive ion exchange chromatography

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4136094A (en) * 1977-08-31 1979-01-23 The Regents Of The University Of Minnesota Preparation of intravenous human and animal gamma globulins and isolation of albumin
US4302384A (en) * 1979-01-17 1981-11-24 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Purification of gammaglobulin derivative
US5118796A (en) * 1987-12-09 1992-06-02 Centocor, Incorporated Efficient large-scale purification of immunoglobulins and derivatives
US5164487A (en) * 1990-03-22 1992-11-17 Biotest Pharma Gmbh Manufacturing intravenous tolerable immunoglobulin-g preparation
US5429746A (en) * 1994-02-22 1995-07-04 Smith Kline Beecham Corporation Antibody purification
US6069236A (en) * 1993-06-14 2000-05-30 Association Pour L'essor De La Transfusion Sanguine Dans La Region Du Nord Immunoglobulin G concentrate for therapeutic use and process for producing said concentrate
US6162904A (en) * 1997-12-24 2000-12-19 Alpha Therapeutic Corporation Manufacturing method for intraveneously administrable immune globulin and resultant product
US6281336B1 (en) * 1998-06-09 2001-08-28 Statens Serum Institut Process for producing immunoglobulins for intravenous administration and other immunoglobulin products
US20020064526A1 (en) * 2000-09-13 2002-05-30 William Pollack Methods of preparing immune globulin and uses thereof
US6441144B1 (en) * 1999-05-20 2002-08-27 Alpha Therapeutic Corporation Method for repairing dual virally inactivated immune globulin for intravenous administration
US20030229212A1 (en) * 2002-04-26 2003-12-11 Robert Fahrner Non-affinity purification of proteins
US20040106780A1 (en) * 1995-05-08 2004-06-03 Suomela Hannu Veli Herman Preparation of immunoglobulin
US20040116674A1 (en) * 1998-06-01 2004-06-17 Genentech, Inc. Separation of polypeptide monomers

Family Cites Families (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0085747B2 (en) 1982-02-08 1990-05-30 Schweizerisches Serum- und Impfinstitut und Institut zur Erforschung der Infektionskrankheiten Intravenously administrable human immunoglobuline and process for its preparation
JPH0582367B2 (en) 1983-08-18 1993-11-18 Nippon Seiyaku Kk
DE3640513C2 (en) 1986-11-27 1990-11-29 Biotest Pharma Gmbh, 6000 Frankfurt, De
US4983722A (en) 1988-06-08 1991-01-08 Miles Inc. Removal of protein A from antibody preparations
US5177194A (en) 1990-02-01 1993-01-05 Baxter International, Inc. Process for purifying immune serum globulins
EP0582595A1 (en) 1991-04-26 1994-02-16 Surface Active Limited Novel antibodies, and methods for their use
DE4118912C1 (en) 1991-06-08 1992-07-02 Biotest Pharma Gmbh, 6072 Dreieich, De
EP0602126B1 (en) 1991-08-14 2003-03-05 Genentech, Inc. Immunoglobulin variants for specific fc epsilon receptors
CA2116246A1 (en) 1992-06-26 1994-01-06 Roland Beliard Human monoclonal anti-rhesus (d) antibodies and cell lines producing same
WO1995016037A1 (en) 1993-12-01 1995-06-15 Menarini Ricerche Sud S.P.A. Anti-egf-r/anti-cd3 bispecific monoclonal antibody, method for its production and its use
ES2129076T5 (en) 1993-12-27 2003-05-16 Zlb Bioplasma Ag Process for the preparation of a concentrated immunoglobulin g anti-d and pharmaceutical composition containing it.
WO1999018130A1 (en) 1997-10-07 1999-04-15 Israel Nur A method for the purification of immunoglobulins
EP0971727A4 (en) 1997-12-24 2005-01-19 Alpha Therapeutic Corp Production process for intravenous immune serum globulin and resultant product
CN101333244B (en) 1998-05-06 2013-12-18 基因技术股份有限公司 Protein purification by ion exchange chromatography
WO1999064462A1 (en) 1998-06-09 1999-12-16 Statens Serum Institut Process for producing immunoglobulins for intravenous administration and other immunoglobulin products
EP1268551B1 (en) 2000-03-30 2004-02-25 Amersham Biosciences AB A METHOD OF PRODUCING IgG
ES2527616T3 (en) * 2002-09-11 2015-01-27 Genentech, Inc. Purification of anti-HER2
CN1490334A (en) 2002-10-18 2004-04-21 缪金明 Affinity purifying method for gene specific antibody
WO2004076485A1 (en) 2003-02-28 2004-09-10 Lonza Biologics Plc. Antibody purification by protein a and ion exchange chromatography
RU2369617C2 (en) 2003-09-10 2009-10-10 Ф. Хоффманн-Ля Рош Аг Antibodies to interleukine-1 receptor and their application
CN101180317B (en) 2005-05-25 2013-06-19 弗·哈夫曼-拉罗切有限公司 Method for the purification of antibodies

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4136094A (en) * 1977-08-31 1979-01-23 The Regents Of The University Of Minnesota Preparation of intravenous human and animal gamma globulins and isolation of albumin
US4302384A (en) * 1979-01-17 1981-11-24 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Purification of gammaglobulin derivative
US5118796A (en) * 1987-12-09 1992-06-02 Centocor, Incorporated Efficient large-scale purification of immunoglobulins and derivatives
US5164487A (en) * 1990-03-22 1992-11-17 Biotest Pharma Gmbh Manufacturing intravenous tolerable immunoglobulin-g preparation
US6069236A (en) * 1993-06-14 2000-05-30 Association Pour L'essor De La Transfusion Sanguine Dans La Region Du Nord Immunoglobulin G concentrate for therapeutic use and process for producing said concentrate
US5429746A (en) * 1994-02-22 1995-07-04 Smith Kline Beecham Corporation Antibody purification
US20040106780A1 (en) * 1995-05-08 2004-06-03 Suomela Hannu Veli Herman Preparation of immunoglobulin
US6162904A (en) * 1997-12-24 2000-12-19 Alpha Therapeutic Corporation Manufacturing method for intraveneously administrable immune globulin and resultant product
US20040116674A1 (en) * 1998-06-01 2004-06-17 Genentech, Inc. Separation of polypeptide monomers
US6281336B1 (en) * 1998-06-09 2001-08-28 Statens Serum Institut Process for producing immunoglobulins for intravenous administration and other immunoglobulin products
US6441144B1 (en) * 1999-05-20 2002-08-27 Alpha Therapeutic Corporation Method for repairing dual virally inactivated immune globulin for intravenous administration
US20020064526A1 (en) * 2000-09-13 2002-05-30 William Pollack Methods of preparing immune globulin and uses thereof
US20030229212A1 (en) * 2002-04-26 2003-12-11 Robert Fahrner Non-affinity purification of proteins
US7323553B2 (en) * 2002-04-26 2008-01-29 Genentech, Inc. Non-affinity purification of proteins

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090118476A1 (en) * 2007-07-17 2009-05-07 Josef Burg Purification of pegylated polypeptides
US20110065903A1 (en) * 2007-07-17 2011-03-17 Josef Burg Chromatographic methods
US20110118448A1 (en) * 2007-07-17 2011-05-19 Josef Burg Purification of pegylated polypeptides
US8138317B2 (en) 2007-07-17 2012-03-20 Hoffmann-La Roche Inc. Purification of pegylated polypeptides
US8795533B2 (en) 2007-07-17 2014-08-05 Hoffmann-La Roche Inc. Chromatographic methods
US8889837B2 (en) 2007-07-17 2014-11-18 Hoffman-La Roche Inc. Purification of pegylated polypeptides
CN103379949A (en) * 2010-10-11 2013-10-30 Abbvie 公司 Processes for purification of proteins

Also Published As

Publication number Publication date Type
KR100967778B1 (en) 2010-07-05 grant
CN101180317B (en) 2013-06-19 grant
ES2389641T3 (en) 2012-10-30 grant
ES2373402T3 (en) 2012-02-03 grant
CA2607375C (en) 2014-01-21 grant
US20140243508A1 (en) 2014-08-28 application
WO2006125599A3 (en) 2007-06-21 application
CA2607375A1 (en) 2006-11-30 application
EP2261252B1 (en) 2012-07-25 grant
JP2008542218A (en) 2008-11-27 application
KR20080017322A (en) 2008-02-26 application
WO2006125599A2 (en) 2006-11-30 application
DK1888636T3 (en) 2011-11-14 grant
US9631007B2 (en) 2017-04-25 grant
US20170183394A1 (en) 2017-06-29 application
EP1888636A2 (en) 2008-02-20 application
CN101180317A (en) 2008-05-14 application
EP2261252A3 (en) 2011-03-23 application
JP4852602B2 (en) 2012-01-11 grant
EP2261252A2 (en) 2010-12-15 application
EP1888636B1 (en) 2011-09-21 grant

Similar Documents

Publication Publication Date Title
US6489447B1 (en) Protein purification
US20030166869A1 (en) Methods for purifying protein
US5429746A (en) Antibody purification
EP1308455A2 (en) A composition comprising anti-HER2 antibodies
US20120238730A1 (en) Integrated approach to the isolation and purification of antibodies
US20050107594A1 (en) Removal of high molecular weight aggregates using hydroxyapatite chromatography
Chen et al. Comparison of standard and new generation hydrophobic interaction chromatography resins in the monoclonal antibody purification process
US20130336957A1 (en) Novel purification of human, humanized, or chimeric antibodies using protein a affinity chromatography
Chen et al. The distinctive separation attributes of mixed-mode resins and their application in monoclonal antibody downstream purification process
WO2004024866A2 (en) Protein purification
US20080177048A1 (en) Enhanced capacity and purification of antibodies by mixed mode chromatography in the presence of aqueous-soluble nonionic organic polymers
US20140288278A1 (en) Chromatography process for resolving heterogeneous antibody aggregates
EP1614693A1 (en) Purification of human monoclonal antibody and human polyclonal antibody
US20140275494A1 (en) Protein purification using displacement chromatography
WO2011110598A1 (en) Method for purifying immunoglobulin solutions
WO2006110277A1 (en) Protein purification using hcic amd ion exchange chromatography
Du et al. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies
WO2006138737A2 (en) Methods of purifying anti a beta antibodies
US20130338344A1 (en) Protein purification methods to reduce acidic species
WO2011009623A1 (en) Optimizing the production of antibodies
US20140072585A1 (en) Novel purification of antibodies using hydrophobic interaction chromatography
US20090186396A1 (en) Enhanced purification of phosphorylated and nonphosphorylated biomolecules by apatite chromatography
WO2011017514A1 (en) Methods for purifying a target protein from one or more impurities in a sample
Azevedo et al. Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography
US20080058507A1 (en) Method For The Removal Of Aggregate Proteins From Recombinant Samples Using Ion Exchange Chromatography

Legal Events

Date Code Title Description
AS Assignment

Owner name: HOFFMANN-LA ROCHE, INC., NEW JERSEY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:F. HOFFMANN-LA ROCHE AG;REEL/FRAME:022356/0681

Effective date: 20071001

Owner name: F. HOFFMANN-LA ROCHE AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FALKENSTEIN, ROBERTO;KOLB, BURKARD;SEBALD, MARIA;REEL/FRAME:022349/0956

Effective date: 20070926