US20110034672A1 - Purification of not-glycosylated polypeptides - Google Patents

Purification of not-glycosylated polypeptides Download PDF

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US20110034672A1
US20110034672A1 US12/811,397 US81139709A US2011034672A1 US 20110034672 A1 US20110034672 A1 US 20110034672A1 US 81139709 A US81139709 A US 81139709A US 2011034672 A1 US2011034672 A1 US 2011034672A1
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chromatography
heterologous polypeptide
glycosylated
hydrophobic
chromatography step
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Roberto Falkenstein
Birgit Weydanz
Nicole Fuehrler
Claudia Giessel
Sybille Greithanner
Adelbert Grossmann
Friederike Hesse
Marc Pompiati
Andreas Schaubmar
Brigitte Kraemer
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Hoffmann La Roche Inc
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Hoffmann La Roche Inc
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Assigned to F. HOFFMANN-LA ROCHE AG reassignment F. HOFFMANN-LA ROCHE AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WEYDANZ, BIRGIT, FALKENSTEIN, ROBERTO, FUEHRLER, NICOLE, GIESSEL, CLAUDIA, GREITHANNER, SYBILLE, GROSSMANN, ADELBERT, HESSE, FRIEDERIKE, KRAEMER, BRIGITTE, POMPIATI, MARC, SCHAUBMAR, ANDREAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

  • the current invention relates to the field of polypeptide purification.
  • a general method for the purification of not-glycosylated polypeptides with a combination of three chromatographic steps is reported.
  • Recombinant polypeptides can be produced e.g. by prokaryotic cells such as E. coli .
  • the recombinantly produced polypeptide accounts for the majority of the prokaryotic cell's polypeptide content and is often deposited as insoluble aggregate, i.e. as a so called inclusion body, within the prokaryotic cell.
  • inclusion bodies For the isolation of the recombinant polypeptide the cells have to be disintegrated and the recombinant polypeptide contained in the inclusion bodies has to be solubilized after the separation of the inclusion bodies from the cell debris.
  • solubilization chaotropic reagents such as urea or guanidinium hydrochloride, are used.
  • reducing agents especially under alkaline conditions, such as dithioerythriol, dithiothreitol, or ⁇ -mercaptoethanol are added.
  • the solubilization of the aggregated polypeptide the globular structure of the recombinant polypeptide, which is essential for the biological activity, has to be reestablished.
  • the concentration of the denaturating agents is slowly reduced, e.g. by dialysis against a suited buffer, which allows the denatured polypeptide to refold into its biologically active structure.
  • the renaturation is the recombinant polypeptide purified to a purity acceptable for the intended use.
  • Recombinantly produced polypeptides obtained from E. coli are normally accompanied by nucleic acids, endotoxins, polypeptides from the producing cell, and not-renaturated recombinant polypeptides.
  • WO 2007/075283 a multi step system and methods of target molecule purification are reported. Methods for purifying compounds comprising a protein of interest are reported in WO 2007/016250. A process for purifying a recombinant protein including one or a few procedural steps only is reported in WO 2006/101441. Rege et al. (Rege, K., Biotechnol. Bioeng. 93 (2006) 618-630) report a high-throughput process development for recombinant protein purification. In KR 2002/080108 a process for purifying human growth hormone from recombinant E. coli is reported.
  • the first aspect of the current invention is a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, wherein the method comprises the following three chromatography steps in the following order:
  • the method according to the invention comprises at least three chromatography steps, whereby for each step a chromatography material can be selected independently of the chromatography material selected for the previous step or for the following step, whereby only the given provisos have to be taken into account.
  • the method according to the invention provides for a flexible and exchangeable sequence of chromatography steps for the purification of a not-glycosylated polypeptide, whereby the obtained purity after subjecting the not-glycosylated polypeptide to the method according to the invention is comparable independently of the selected chromatography step sequence.
  • the prokaryotic cell an E. coli cell.
  • the affinity chromatography a metal chelating chromatography.
  • the method comprises the method an additional step either after step a) or after step b) or after step c) which is d) PEGylating said polypeptide.
  • said steps a) and b) are cation exchange chromatography.
  • a second aspect of the current invention is a method for the recombinant production of a not-glycosylated heterologous polypeptide in a prokaryotic cell, wherein the method comprises the following steps:
  • the methods according to the invention are characterized in that at least two different sequences of three chromatographic steps yield a purified not-glycosylated, heterologous polypeptide with comparable purity.
  • the third chromatography step can be performed in flow-through mode with polypeptides having a low, i.e. 6.0 or lower, or high, i.e. 8.0 or higher, isoelectric point.
  • Methods for purifying polypeptides are well established and widespread used. They are employed either alone or in combination. Such methods are, for example, affinity chromatography using thiol ligands with complexed metal ions (e.g. with Ni(II)- and Cu(II)-affinity material) or microbial-derived 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 chromatography), thiophilic adsorption (e.g. with beta-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g.
  • buffer substance denotes a solution in which changes of pH due to the addition or release of acidic or basic substances is leveled by a buffer substance. Any buffer substance resulting in such an effect can be used.
  • pharmaceutically acceptable buffer substances are used, such as e.g. phosphoric acid or salts thereof, acetic acid or salts thereof, citric acid or salts thereof, morpholine or salts thereof, 2-(N-morpholino) ethanesulfonic acid or salts thereof, histidine or salts thereof, glycine or salts thereof, or Tris (hydroxymethyl) aminomethane (TRIS) or salts thereof.
  • the buffered solution may comprise an additional salt, such as e.g. sodium chloride, sodium sulphate, potassium chloride, potassium sulfate, sodium citrate, or potassium citrate.
  • membrane denotes both a microporous or macroporous membrane.
  • the membrane itself is composed of a polymeric material such as, e.g. polyethylene, polypropylene, ethylene vinyl acetate copolymers, polytetrafluoroethylene, polycarbonate, poly vinyl chloride, polyamides (nylon, e.g.
  • ZetaporeTM, N 66 PosidyneTM polyesters, cellulose acetate, regenerated cellulose, cellulose composites, polysulphones, polyethersulfones, polyarylsulphones, polyphenylsulphones, polyacrylonitrile, polyvinylidene fluoride, non-woven and woven fabrics (e.g. Tyvek®), fibrous material, or of inorganic material such as zeolithe, SiO 2 , Al 2 O 3 , TiO 2 , or hydroxylapatite.
  • Tyvek® non-woven and woven fabrics
  • chromatography material denotes on the one hand a solid material that can be used without further modification as chromatography material, such as hydroxylapatite or affinity chromatography material, and also material comprising a bulk core material to which chromatographical functional groups are attached, preferably by covalent bonds.
  • the bulk core material is understood to be not involved in the chromatography process, i.e. the interaction between the polypeptide to be separated and the chromatographical functional groups of the chromatography material. It is merely providing a three dimensional framework to which the chromatographical functional groups are attached and which ensures that the solution containing the substance to be separated can access the chromatographical functional group.
  • said bulk core material is a solid phase.
  • said “chromatography material” is a solid phase to which chromatographical functional groups are attached, preferably by covalent bonds.
  • said “chromatographical functional group” is an ionizable hydrophobic group, or a hydrophobic group, or a complex group in which different chromatographical functional groups are combined in order to bind only a certain type of polypeptide, or a covalently bound charged group.
  • a “solid phase” denotes a non-fluid substance, and includes particles (including microparticles and beads) made from materials such as polymer, metal (paramagnetic, ferromagnetic particles), glass, and ceramic; gel substances such as silica, alumina, and polymer gels; zeolites and other porous substances.
  • a solid phase may be a stationary component, such as a packed chromatography column, or may be a non-stationary component, such as beads and microparticles.
  • Such particles include polymer particles such as polystyrene and poly(methylmethacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See for example Martin, C. R., et al., Analytical Chemistry-News & Features, May 1 (1998) 322A-327A.
  • hydrophobic charge induction chromatography or “HCIC”, which can be used interchangeably within this application, denote a chromatography method which employs a “hydrophobic charge induction chromatography material”.
  • a “hydrophobic charge induction chromatography material” is a chromatography material which comprises chromatographical function groups which can in one pH range form hydrophobic bonds to the substance to be separated and which are charged either positively or negatively in other pH ranges, i.e. HCIC uses ionizable hydrophobic groups as chromatographical functional group.
  • HCIC uses ionizable hydrophobic groups as chromatographical functional group.
  • the polypeptide is bound to the hydrophobic charge induction material under neutral pH conditions and recovered afterwards by the generation of charge repulsion by a change of the pH value.
  • An exemplary “hydrophobic charge induction chromatography materials” is BioSepra MEP or HEA Hypercel (Pall Corp., USA).
  • hydrophobic interaction chromatography or “HIC”, which can be used interchangeably within this application, denote a chromatography method in which a “hydrophobic interaction chromatography material” is employed.
  • a “hydrophobic interaction chromatography material” is a chromatography material to which hydrophobic groups, such as butyl-, octyl-, or phenyl-groups, are bound as chromatographical functional groups.
  • the polypeptides are separated depending on the hydrophobicity of their surface exposed amino acid side chains, which can interact with the hydrophobic groups of the hydrophobic interaction chromatography material.
  • the interactions between polypeptides and the chromatography material can be influenced by temperature, solvent, and ionic strength of the solvent.
  • a temperature increase e.g. supports the interaction between the polypeptide and the hydrophobic interaction chromatography material as the motion of the amino acid side chains increases and hydrophobic amino acid side chains buried inside the polypeptide at lower temperatures become accessible. Also is the hydrophobic interaction promoted by kosmotropic salts and decreased by chaotropic salts.
  • “Hydrophobic interaction chromatography materials” are e.g. Phenylsepharose CL-4B, 6 FF, HP, Phenyl Superose, Octylsepharose CL-4B, 4 FF, and Butylsepharose 4 FF (all available from Amersham Pharmacia Biotech Europe GmbH, Germany), which are obtained via glycidyl-ether coupling to the bulk material.
  • affinity chromatography denotes a chromatography method which employs an “affinity chromatography material”.
  • affinity chromatography the polypeptides are separated based on their biological activity or chemical structure depending of the formation of electrostatic interactions, hydrophobic bonds, and/or hydrogen bond formation to the chromatographical functional group.
  • a competitor ligand is added or the chromatography conditions, such as pH value, polarity or ionic strength of the buffer are changed.
  • An “affinity chromatography material” is a chromatography material which comprises a complex chromatographical functional group in which different single chromatographical functional groups are combined in order to bind only a certain type of polypeptide.
  • affinity chromatographical materials are a “metal chelating chromatography material” such as Ni(II)-NTA or Cu(II)-NTA containing materials, for the binding of fusion polypeptides containing a hexahistidine tag or polypeptides with a multitude of surface exposed histidine, cysteine, and/or tryptophane residues, or an “antibody binding chromatography material” such a protein A, or an “enzyme binding chromatography material” such as chromatography materials comprising enzyme substrate analogues, enzyme cofactors, or enzyme inhibitors as chromatographical functional group, or a “lectin binding chromatography material” such as chromatography materials comprising polysaccharides, cell surface receptors, glycoproteins, or intact cells as chromatographical functional group.
  • metal chelating chromatography denotes a chromatography method which employs a “metal chelating chromatography material”.
  • Metal chelating chromatography is based on the formation of chelates between a metal ion, such as Cu(II), Ni(II) or Zn(II), which is bound to a bulk material as chromatographical functional groups, and electron donor groups of surface exposed amino acid side chains of polypeptides, especially with imidazole containing side chains and thiol group containing side chains.
  • the chelate is formed at pH values at which those side chains are at least partly not protonated.
  • the bound polypeptide is recovered from the chromatography material by a change in the pH value, i.e. by protonation.
  • Exemplary “metal chelating chromatography materials” are HiTrap Chelating HP (Amersham Pharmacia Biotec Europe GmbH, Germany), or Fraktogel EMD (EMD Chemicals Inc, USA).
  • ion exchange chromatography denotes a chromatography method which employs an “ion exchange chromatography material”.
  • ion exchange chromatography material encompasses depending whether a cation is exchanged in a “cation exchange chromatography” a “cation exchange chromatography material” or an anion is exchanged in an “anion exchange chromatography” an “anion exchange chromatography material”.
  • ion exchange chromatography material denotes an immobile high molecular weight solid phase that carries covalently bound charged groups as chromatographical functional groups. For overall charge neutrality not covalently bound counter ions are associated therewith.
  • the “ion exchange chromatography material” has the ability to exchange its not covalently bound counter ions for similarly charged ions of the surrounding solution.
  • the “ion exchange chromatography material” is referred to as “cation exchange chromatography material” or as “anion exchange chromatography material”.
  • the “ion exchange chromatography material” is referred to as e.g. in the case of cation exchange chromatography materials with sulfonic acid groups (S), or carboxymethyl groups (CM).
  • S sulfonic acid groups
  • CM carboxymethyl groups
  • the “ion exchange chromatography material” can additionally be classified as strong or weak ion exchange chromatography material, depending on the strength of the covalently bound charged substituent.
  • strong cation exchange chromatography materials have a sulfonic acid group as chromatographical functional group and weak cation exchange chromatography materials have a carboxylic acid group as chromatographical functional group.
  • “Cation exchange chromatography materials”, for example, 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-SepharoseTM Fast Flow (available from GE Healthcare—Amersham Biosciences Europe GmbH, Freiburg, Germany).
  • hydroxylapatite chromatography denotes a chromatography method that employs a certain form of calcium phosphate as chromatography material.
  • Exemplary hydroxylapatite chromatography materials are Bio-Gel HT, Bio-Gel HTP, Macro-Prep Ceramic (available from Biorad Laboratories), Hydroxylapatite Type I, Type II, HA Ultrogel (Sigma Aldrich Chemical Corp., USA), Hydroxylapatite Fast Flow and High Resolution (Calbiochem), or TSK gel HA-1000 (Tosoh Haas Corp., USA)
  • 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.”
  • a “protein” is a molecule comprising one or more polypeptide chains whereof at least one comprises 100 or more amino acid residues. Polypeptides and protein may also comprise non-amino acid components, such as carbohydrate groups. Carbohydrate groups and other non-amino acid components may be added by the cell in which the polypeptide or protein is produced, and will vary with the type of cell. Polypeptides and 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.
  • antibody and “immunoglobulin”, which can be used interchangeably within this application, denote a molecule generally comprising two light chains and two heavy chains.
  • Each of the heavy and light chains comprises a variable region (generally the amino terminal portion of the chain) which contains specific binding regions (CDR, complementary determining region) which interacts with the antigen.
  • CDR specific binding regions
  • Each of the heavy and light chains also comprises a constant region (generally, the carboxyl terminal portion of the chains) which may mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system, some phagocytic cells and a first component (Clq) of the classical complement system.
  • the light and heavy chains of an immunoglobulin are complete chains, each consisting essentially of a variable region and a complete constant region.
  • a light chain comprises a light chain variable domain, a hinge region, and a light chain constant domain
  • a heavy chain comprises a heavy chain variable domain, a hinge region, and a heavy chain constant domain consisting of a C H 1 domain, a C H 2 domain, a C H 3 domain, and optionally a C H 4 domain.
  • 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., Proc. Natl. Acad. Sci.
  • immunoglobulins Depending on the amino acid sequence of the constant region of the heavy chain are immunoglobulins assigned to different classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), i.e. IgG in IgG 1, IgG2, IgG3, and IgG4, or IgA in IgA1 and IgA2.
  • immunoglobulin class to which an immunoglobulin belongs the heavy chain constant regions of immunoglobulins are called ⁇ (IgA), ⁇ (IgD), ⁇ (IgE), ⁇ (IgG), and ⁇ (IgM), respectively.
  • binding-and-elute mode denotes an operation mode of a chromatography method, in which a solution containing a substance of interest is brought in contact with a stationary phase, preferably a solid phase, whereby the substance of interest binds to the stationary phase.
  • a stationary phase preferably a solid phase
  • the substance of interest is afterwards eluted from the stationary phase in a second step and thereby recovered from the stationary phase with an elution solution.
  • This does not necessarily denote that 100% of the substances not of interest are removed but essentially 100% of the substances not of interest are removed, i.e. at least 50% of the substances not of interest are removed, preferably at least 75% of the substances not of interest are removed, preferably at least 90% of the substances not of interest are removed, preferably more than 95% of the substances not of interest are removed.
  • flow-through mode denotes an operation mode of a chromatography method, in which a solution containing a substance of interest is brought in contact with a stationary phase, preferably a solid phase, whereby the substance of interest does not bind to that stationary phase.
  • a stationary phase preferably a solid phase
  • the substance of interest is obtained either in the flow-through or the supernatant.
  • Substances not of interest, which were also present in the solution bind to the stationary phase and are removed from the solution. This does not necessarily denote that 100% of the substances not of interest are removed from the solution but essentially 100% of the substances not of interest are removed, i.e.
  • At least 50% of the substances not of interest are removed from the solution, preferably at least 75% of the substances not of interest are removed from the solution, preferably at least 90% of the substances not of interest are removed from the solution, preferably more than 95% of the substances not of interest are removed from the solution.
  • continuous elution and “continuous elution method”, which are used interchangeably within this application, denote a chromatography method wherein e.g. the concentration of a substance causing elution, i.e. the dissolution of a bound substance from a chromatography material, is raised or lowered continuously, i.e. the concentration is changed by a sequence of small steps each not bigger than a change of 2%, preferably of 1%, of the concentration of the substance causing elution.
  • one or more conditions for example the pH, the ionic strength, concentration of a salt, and/or the flow of a chromatography method, may be changed linearly, or changed exponentially, or changed asymptotically. Preferably the change is linear.
  • step elution and “step elution method”, which are used interchangeably within this application, denote a chromatography method wherein e.g. the concentration of a substance causing elution, i.e. the dissolution of a bound substance from a chromatography material, is raised or lowered at once, i.e. directly from one value/level to the next value/level.
  • concentration of a substance causing elution i.e. the dissolution of a bound substance from a chromatography material
  • step elution one or more conditions, for example the pH, the ionic strength, concentration of a salt, and/or the flow of a chromatography method, is/are changed all at once from a first, e.g. starting, value to a second, e.g. final, value.
  • Step elution denotes that the conditions are changed incrementally, i.e. stepwise, in contrast to a linear change.
  • step elution method is after each increase a new fraction collected. After each increase the conditions are maintained till the next step in the elution method.
  • an “isolated polypeptide” is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of an isolated polypeptide contains the polypeptide in a highly purified form, i.e. at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers, derivatized forms, not correctly folded forms, not correctly disulfide bridged forms, or scrambled forms.
  • Heterologous DNA refers to a DNA molecule or a polypeptide, or a population of DNA molecules or a population of polypeptides that do not exist naturally within a given cell.
  • DNA molecules heterologous to a particular cell may contain DNA derived from the cell's species (i.e. endogenous DNA) so long as that cell's DNA is combined with non-cell's DNA (i.e. exogenous DNA).
  • endogenous DNA DNA i.e. exogenous DNA
  • a DNA molecule containing a non-cell's DNA segment encoding a polypeptide operably linked to a cell's DNA segment comprising a promoter is considered to be a heterologous DNA molecule.
  • heterologous DNA molecule can comprise an endogenous structural gene operably linked with an exogenous promoter.
  • a peptide or polypeptide encoded by a non-cell's DNA molecule is a “heterologous” peptide or polypeptide.
  • the current invention provides in a first aspect a method for purifying a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, comprising the following steps in the following order:
  • the purified not-glycosylated, heterologous polypeptide obtained after step c) of the method according to the invention is the purified not-glycosylated, heterologous polypeptide obtained after step c) of the method according to the invention. Due to the different characteristics of different polypeptides, which are depending on its physical properties, such as e.g. the isoelectric point (Ip) or the distribution of surface exposed amino acid residues, not all chromatography methods are suited for all polypeptides. Therefore the following provisos apply to the method according to the invention:
  • a first exemplary polypeptide is an IGF-1 agonist as reported e.g. in WO 2006/066891.
  • the sequence comprises the chromatography steps:
  • This sequence fulfills the provisos for the method according to the invention as the polypeptide has a hexahistidine tag and an isoelectric point above 6.0.
  • the starting material had a purity of 50% (determined by HPLC) of the IGF-1 agonist.
  • a purity of more than 97% (determined by HPLC) has been obtained. All three chromatography steps have been performed in a bind-and-elute mode.
  • the IGF-1 agonist has also been purified with a different sequence of chromatography steps according to the method according to the invention which are:
  • the same molecule can be purified to a similar purity.
  • the final chromatography step can be performed in different elution modes, i.e. in a bind-and-elute mode or in a flow-through mode.
  • the different chromatography steps can be performed either as step elution or as continuous elution.
  • another aspect of the current invention is a method for the purification of a polypeptide, especially of IGF-1 or an IGF-1 variant as reported in WO 2006/066891, comprising a sequence of three successive chromatography steps whereby the first chromatography step is a hydrophobic charge induction chromatography, the second chromatography step is selected from hydroxylapatite chromatography or cation exchange chromatography, and the third chromatography step is selected from hydrophobic charge induction chromatography or anion exchange chromatography.
  • a second exemplary polypeptide is interferon alpha-2a (IFN ⁇ -2a) as reported e.g. in EP 0 043 980.
  • the sequence comprises the chromatography steps:
  • This sequence fulfills the provisos for the method according to the invention as the recombinantly produced IFN ⁇ -2a has no tag for the interaction with a metal chelating chromatography material and has an isoelectric point above 6.0.
  • the starting material had a purity of 49% (determined by HPLC). After performing the purification method according to the invention with the chromatography steps as outlined above a purity of more than 99% (determined by HPLC) has been obtained. The chromatography steps have been performed in a bind-and-elute mode.
  • another aspect of the current invention is a method for the purification of IFN ⁇ -2a comprising a sequence of three successive chromatography steps, whereby the first chromatography step is a hydrophobic charge induction chromatography step, the second chromatography step is an anion exchange chromatography step, and the third chromatography step is an hydrophobic charge induction chromatography step.
  • the IFN ⁇ -2a has also been purified for comparison with a different sequence of chromatography steps:
  • another aspect of the current invention is a method for the purification of IFN ⁇ -2a comprising a sequence of three successive chromatography steps, whereby the first chromatography step is a hydrophobic interaction chromatography, the second chromatography step is a cation exchange chromatography step, and the third chromatography step is an hydrophobic interaction chromatography.
  • the method according to the invention is not only applicable to not-glycosylated, recombinantly produced polypeptides, it is further more also suitable for the production of PEGylated, not-glycosylated polypeptides.
  • PEGylated interferon see e.g. EP 0 809 996.
  • Another aspect of the current invention is a method for producing a not-glycosylated, PEGylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell comprising the following steps in the following order:
  • the starting material had a purity of 58% (determined by HPLC). After performing the purification method according to the invention with the chromatography steps as outlined above a purity of more than 90% (determined by HPLC) has been obtained. All the chromatography steps have been performed in a bind-and-elute mode.
  • the IFN has also been purified prior to PEGylation with a further sequence of chromatography steps according to the method according to the invention:
  • another aspect of the current invention is a method for the production of a PEGylated IFN ⁇ -2a comprising a sequence of three successive chromatography steps whereby the first chromatography step is selected from hydrophobic interaction chromatography or metal affinity chromatography, the second chromatography step is a cation exchange chromatography, and the third chromatography step is an anion exchange chromatography and wherein after the third chromatography step the purified not-glycosylated and not-PEGylated IFN is PEGylated.
  • another aspect of the current invention is a method for the production of PEGylated interferon, especially IFN ⁇ -2a, comprising a sequence of three successive chromatography steps whereby the first chromatography step is hydrophobic interaction chromatography, the second chromatography step is a cation exchange chromatography, and the third chromatography step is an hydrophobic charge induction chromatography and wherein after the third chromatography step the purified not-glycosylated and not-PEGylated IFN is PEGylated.
  • Escherichia, Salmonella, Streptomyces or Bacillus are, for example, suitable as prokaryotic host organisms.
  • the prokaryotic cell an E. coli cell.
  • the E. coli cell is an E. coli XL1-blue cell, or an E. coli BL21(DE3) cell, or an E. coli K-12 cell.
  • Another aspect of the current invention is a method for the recombinant production of a not-glycosylated heterologous polypeptide in a prokaryotic cell, characterized in that said method comprises the following steps:
  • the not-glycosylated heterologous polypeptide obtained after step c is the not-glycosylated heterologous polypeptide obtained after step c). Due to the different characteristics of different polypeptides, which are depending on its physical properties, such as e.g. the isoelectric point (Ip) or the distribution of surface exposed amino acid residues, not all chromatography methods are suited for all polypeptides. Therefore the following provisos apply to the method according to the invention:
  • under conditions suitable denotes conditions which are used for the cultivation of a cell expressing a polypeptide and which are known to or can easily be determined by a person skilled in the art. It is known to a person skilled in the art that these conditions may vary depending on the type of cell cultivated and type of polypeptide expressed. In general the cell is cultivated at a temperature, e.g. between 20° C. and 40° C., and for a period of time sufficient to allow effective production, e.g. for of from 4 to 28 days.
  • bind and elute mode denotes an operation mode of a purification method, in which a solution containing a substance of interest to be purified is brought in contact with a stationary phase, preferably a solid phase, whereby the substance of interest binds to the stationary phase.
  • a stationary phase preferably a solid phase
  • the substance of interest is afterwards optionally after a washing step eluted from the stationary phase in a second step and thereby recovered from the stationary phase with an elution solution.
  • PEGylating means the formation of a covalent linkage of a (polyethylene) glycol residue at the N-terminus of the polypeptide and/or an internal lysine residue. PEGylation of proteins is widely known in the state of the art and reviewed by, for example, Veronese, F. M., Biomaterials 22 (2001) 405-417. PEG can be linked using different functional groups and polyethylene glycols with different molecular weight, linear and branched PEGs as well as different linking groups (see also Francis, G. E., et al., Int. J. Hematol. 68 (1998) 1-18; Delgado, C., et al., Crit. Rev. Ther.
  • PEG derivatives are known in the art and are described in, for example, Morpurgo, M., et al., J. Bioconjug. Chem. 7 (1996) 363-368, for PEG-vinylsulfone. Linear chain and branched chain PEG species are suitable for the preparation of the PEGylated fragments. Examples of reactive PEG reagents are iodo-acetyl-methoxy-PEG, or methoxy-PEG-vinylsulfone.
  • the methods according to the current invention is the content of endotoxins, and/or E. coli DNA, and/or E. coli cell proteins reduced in the polypeptide solution obtained after the third chromatography step compared to the content prior to the first chromatography step.
  • the method according to the invention a method for the recombinant production of a not-glycosylated heterologous polypeptide in a prokaryotic cell via inclusion bodies, whereby the method comprises the following steps:
  • In one embodiment is the not-glycosylated heterologous polypeptide obtained after step d).
  • Inclusion bodies are found in the cytoplasm and contain the expressed polypeptide in an aggregated form insoluble in water. Usually, such proteins of inclusion bodies are in a denatured form (e.g., randomly linked disulfide bridges). These inclusion bodies are separated from other cell components, for example by centrifugation after cell lysis. According to the invention, the inclusion bodies are washed under denaturing conditions.
  • denaturing agents are well known in the state of the art and are, for example, highly concentrated solutions of guanidinium hydrochloride (e.g. about 6 mol/l) or urea (e.g. about 8 mol/l). The denaturing agent is preferably used as a buffered solution. After washing, the inclusion bodies are solubilized.
  • PEGylation means a covalent linkage of a poly (ethylene glycol) residue at the N-terminus of the polypeptide and/or an internal lysine residue.
  • PEGylation of proteins is widely known in the state of the art and reviewed by, for example, Veronese, F. M., Biomaterials 22 (2001) 405-417.
  • PEG can be linked using different functional groups and polyethylene glycols with different molecular weight, linear and branched PEGs as well as different linking groups (see also Francis, G. E., et al., Int. J. Hematol. 68 (1998) 1-18; Delgado, C., et al., Crit. Rev. Ther.
  • PEGylation can be performed in aqueous solution with PEGylation reagents as described, for example, in WO 00/44785, in one embodiment by using NHS-activated linear or branched PEG molecules of a molecular weight between 5 kDa and 40 kDa. PEGylation can also be performed at the solid phase according to Lu, Y., et al., Reactive Polymers 22 (1994) 221-229. Not randomly, N-terminally PEGylated polypeptide can also be produced according to WO 94/01451.
  • Activated PEG derivatives are known in the art and are described in, for example, Morpurgo, M., et al., J. Bioconjug. Chem. 7 (1996) 363-368, for PEG-vinylsulfone. Linear chain and branched chain PEG species are suitable for the preparation of the PEGylated fragments.
  • the PEG species an activated PEG ester, e.g., N-hydroxysuccinimidyl propionate, or N-hydroxysuccinimidyl butanoate, or N-hydroxysuccinimides such as PEG-NHS (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).
  • the activated N-hydroxysuccinimide ester is
  • alkoxy-PEG-N-hydroxysuccinimide such as methoxy-PEG-N-hydroxysuccinimide (MW 30000; Shearwater Polymers, Inc.), wherein R and m are as defined above.
  • the PEG species is the N-hydroxysuccinimidyl ester of methoxy poly (ethylene glycol)-butyric acid.
  • alkoxy refers to an alkyl ether group in which the term ‘alkyl’ means a straight-chain or branched-chain alkyl group containing a maximum of four carbon atoms, such as methoxy, ethoxy, n-propoxy and the like, preferably methoxy.
  • One aspect of the invention is a method for the purification of a not-glycosylated, heterologous polypeptide, which has been recombinantly produced in a prokaryotic cell, characterized in that said method comprises the following steps in the following order:
  • the method according to the invention is the not-glycosylated heterologous polypeptide obtained after the third chromatography step.
  • FIG. 1 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the first HCIC.
  • FIG. 2 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the hydroxylapatite chromatography step.
  • FIG. 3 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the second HCIC.
  • FIG. 4 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the HIC.
  • FIG. 5 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the cation exchange chromatography step.
  • FIG. 6 Reversed Phase HPLC chromatogram of the IGF-1 agonist before (a) and after (b) the anion exchange chromatography step.
  • Protein concentration was determined by determining the optical density (OD) at 280 nm, with a reference wavelength of 320 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence.
  • the chromatography was conducted with a Tosoh Haas TSK 3000 SWXL column on an ASI-100 HPLC system (Dionex, Idstein, Germany). The elution peaks were monitored at 280 nm by a UV diode array detector (Dionex). After dissolution of the concentrated samples to 1 mg/ml the column was washed with a buffer consisting of 200 mM potassium dihydrogen phosphate and 250 mM potassium chloride pH 7.0 until a stable baseline was achieved. The analyzing runs were performed under isocratic conditions using a flow rate of 0.5 ml/min. over 30 minutes at room temperature. The chromatograms were integrated manually with Chromeleon (Dionex, Idstein, Germany).
  • the purity is analyzed by RP-HPLC.
  • the assay is performed on a Poroshell column using an acetonitrile/aqueous TFA gradient.
  • the elution profile is monitored as UV absorbance at 215 nm.
  • the percentages of the eluted substances are calculated based upon the total peak area of the eluted proteins.
  • 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.
  • 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.
  • 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.
  • inclusion bodies e.g. be performed according the method by Rudolph et al. (Rudolph et al., Folding Proteins, In: T. E. Creighton (ed.): Protein function: A Practical Approach, 57 (1996)).
  • the inclusion bodies were stored at ⁇ 70° C. Solubilization of the inclusion bodies can likewise be performed according the method by Rudolph et al. (Rudolph et al., Folding Proteins, In: T. E. Creighton (ed.): Protein function: A Practical Approach, 57 (1996)).
  • the polypeptide was expressed in E. coli .
  • the polypeptide is first applied to a HCIC column, then to a hydroxylapatite column and finally to a second HCIC column.
  • the solution containing the IGF-1 agonist was applied in a first step to a column containing a hydrophobic charge induction chromatography material (MEP-Hypercel from Pall Corporation).
  • FIG. 1 the reversed phase chromatogram of the IGF-1 agonist before and after HCIC is presented.
  • FIG. 2 presents the reversed phase chromatogram before and after the hydroxylapatite chromatography step.
  • FIG. 3 presents the reversed phase chromatogram before and after the second HCIC step.
  • the polypeptide is first applied to a HIC column, followed by a cation exchange chromatography and finally to an anion exchange chromatography operated in flow-through mode.
  • Elution was performed with a linear gradient over 30 column volumes from 0% (v/v) to 100% (v/v) of buffer B.
  • FIG. 4 presents the reversed phase chromatogram before and after the HIC step.
  • Elution was performed as follows: change to 15% (v/v) buffer B at the start, maintaining 15% (v/v) buffer B for 5 column volumes, afterwards a linear gradient to 55% (v/v) buffer B over 20 column volumes, and finally maintaining 55% (v/v) buffer B for 10 column volumes.
  • FIG. 5 presents the reversed phase chromatogram before and after the cation exchange chromatography step.
  • FIG. 6 presents the reversed phase chromatogram before and after the anion exchange chromatography step.
  • the polypeptide is first applied to a HIC column, then to an anion exchange column and finally to a cation exchange column.
  • Elution was performed as follows: change to 15% (v/v) buffer B at the start, maintaining 15% (v/v) buffer B for 3 column volumes, and afterwards a linear gradient to 90% (v/v) buffer B over 37.5 column volumes.
  • the polypeptide is first applied to a HIC column, then to a cation exchange column and finally to an anion exchange column.
  • the polypeptide is first applied to a metal chelating column, then to a cation exchange column and finally to an anion exchange column.
  • Resin Anion exchange chromatography with Q-Sepharose Loading: 3 mg polypeptide per ml of column volume Buffer A: 30 mM ammonium acetate buffer, adjusted to pH 6.8 Buffer B: 1) 25 mM ammonium acetate, adjusted to pH 6.5
  • Elution was performed as follows: change to 10% (v/v) buffer B at the start, maintaining 15% (v/v) buffer B for 3 column volumes, and afterwards a linear gradient to 90% (v/v) buffer B over 27.5 column volumes.

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