WO2011003600A1 - Procédé de purification d'interféron-bêta - Google Patents

Procédé de purification d'interféron-bêta Download PDF

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WO2011003600A1
WO2011003600A1 PCT/EP2010/004130 EP2010004130W WO2011003600A1 WO 2011003600 A1 WO2011003600 A1 WO 2011003600A1 EP 2010004130 W EP2010004130 W EP 2010004130W WO 2011003600 A1 WO2011003600 A1 WO 2011003600A1
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ifn
chromatography
affinity chromatography
formulation
purification
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PCT/EP2010/004130
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WO2011003600A8 (fr
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Stefan Arnold
Christian Scheckermann
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Biogenerix Ag
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Priority to US13/382,807 priority Critical patent/US20120177603A1/en
Priority to EP10737494A priority patent/EP2451470A1/fr
Priority to JP2012518819A priority patent/JP2012532167A/ja
Priority to EA201290040A priority patent/EA201290040A1/ru
Publication of WO2011003600A1 publication Critical patent/WO2011003600A1/fr
Publication of WO2011003600A8 publication Critical patent/WO2011003600A8/fr

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    • 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]
    • C07K14/565—IFN-beta
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for the production of recombinant human Interferon- ⁇ (IFN- ⁇ ), comprising at least one affinity chromatography (AC) and at least one hydrophobic interaction chromatography (HIC) step.
  • IFN- ⁇ recombinant human Interferon- ⁇
  • AC affinity chromatography
  • HIC hydrophobic interaction chromatography
  • the present invention relates to a method for the purification of glycosylated IFN- ⁇ from cell culture supernatant or a mixture of other proteins, comprising two affinity chromatography steps with subsequent hydrophobic interaction chromatography steps as preferably followed by an anion exchange chromatography (AEX) step.
  • AEX anion exchange chromatography
  • Interferons are species specific proteins, partially glycoproteins, which are secreted by different cell types of the body upon induction by viruses, double-stranded RNA, other polynucleotides as well as antigens. Interferons possess numerous biological activities such as antiviral, antiproliferative as well as immunomodulating properties. So far, at least three different types of human interferons have been identified, which are produced by leucocytes, lymphocytes, fibroblasts as well as cells of the immune system and designated ⁇ -, ⁇ -, and ⁇ -interferons. Several interferon types are further subdivided in subtypes.
  • Native human IFN- ⁇ can be industrially produced by superinduction of human fibroblast cell cultures with PoIy-IC and subsequent isolation and purification of IFN- ⁇ by chromatographic and electrophoretic techniques. Applying recombinant DNA technology, proteins or polypeptides having comparable features as naturally occurring IFN- ⁇ can be produced; see for example European patent applications EP 028 033, EP 0 041 313, EP 070 906 and EP 287 075 as well as Chemajovsky et al. DNA 3 (1984), 297-308 and McCormick et al. MoI. Cell. Biol. 4 (1984), 166-172.
  • human recombinant IFN- ⁇ can be produced by eukaryotic cells (for example CHO cells) or by prokaryotic cells (for example E.coli).
  • the corresponding interferons are designated IFN- ⁇ - Ia and IFN- ⁇ - Ib, respectively.
  • IFN- ⁇ -la is glycosylated; see Goodkin, Lancet 344 (1994), 1057-1060.
  • Interferon- ⁇ A prerequisite tor the therapeutic application of Interferon- ⁇ implies that it can be provided in sufficient amounts and high purity and it is formulated in a galenic composition, which makes the protein suitable for long-term storage by maintaining the molecular integrity. Interferon- ⁇ is instable and is subject to different degradation reactions. These include especially the cleavage of peptide bonds, deamidation, oxidation of methionine to methioninsulfide, disulfide exchange as well as modification of the sugar side chain up to deglycosylation.
  • Murine Interferon- ⁇ differs significantly from human IFN- ⁇ . Therefore, the principles for purifying murine IFN- ⁇ described in the numerous available literature are not transferable and in the last decade, intensive efforts have been made to provide and optimize purification protocols for IFN- ⁇ , in order to isolate IFN- ⁇ in a sufficient amount and in a form in which it is suitable for stable storage and therapeutic use. Therefore, several publications exist, which describe different purification methods of IFN- ⁇ .
  • European patent application EP 011 435 discloses a sequence of purification steps comprising a cation exchange and a metal chelate affinity chromatography step.
  • European patent application EP 027 262 describes a method for purification comprising a dye ligand chromatography with Cibacron Blue.
  • European patent application EP 041 313 describes the use of a zinc chelate chromatography for purification of IFN- ⁇ .
  • European patent application EP 094 672 and EP 118 808 describe a purification method comprising Cibacron Blue and metal chelate chromatography.
  • European patent application EP 215 658 describes a sequence of purification steps comprising an affinity chromatography and a High Performance Liquid Chromatography (HPLC).
  • HPLC High Performance Liquid Chromatography
  • EP 274 900 the purification of IFN- ⁇ inter alia by affinity chromatography and reverse phase HPLC (RP-HPLC) is described.
  • RP-HPLC reverse phase HPLC
  • EP 467 992 describes the use of a metal chelate chromatography for the purification of IFN- ⁇ .
  • European patent application EP 529 300 discloses a sequence of purification steps comprising liquid/liquid phase extraction, Cibacron Blue affinity chromatography, immobilized Metal Ion Affinity Chromatography (IMAC) and gel chromatography.
  • IMAC immobilized Metal Ion Affinity Chromatography
  • German patent application DE 30 28 919 describes a sequence of purification steps comprising affinity chromatography and RP-HPLC.
  • German patent application DE 30 39 566 the purification of IFN- ⁇ inter alia by glass absorption and a metal chelate chromatography step is described.
  • European patent application EP 446 850 describes a sequence of purification steps comprising glass absorption and cation exchange chromatography (CEX).
  • IFN- ⁇ immunoaffinity chromatography
  • lectin affinity chromatography in particular by use of Concanavalin A (ConA)
  • ConA Concanavalin A
  • Phenyl- Sepharose has been described; see for example Carter and Horoszewicz, Pharmacol. Ther. 8 (1980), 359-377 as well as Mikulski et al. Prep. Biochem. 10 (1980), 103-1 19.
  • the object of the present invention is to provide a method for purifying biologically active recombinant human IFN- ⁇ in satisfactory purity and amount. Furthermore, the method should be simple and straightforward in realization. Desirable is a purification process which is applicable in routine process under GMP (Good Manufacturing Practice) aspects, and which preferably takes requirements for regulatory acceptance (validation, reproducibility) and particular biochemical peculiarities (such hydrophobicity) of IFN- ⁇ into account.
  • GMP Good Manufacturing Practice
  • the present invention relates to a method for purification of recombinantly produced biologically active human IFN- ⁇ (IFN- ⁇ ), comprising at least one affinity chromatography (AC) step and at least one hydrophobic interaction chromatography (HIC) step, wherein these chromatography steps can be performed immediately after another in either order.
  • IFN- ⁇ biologically active human IFN- ⁇
  • AC affinity chromatography
  • HIC hydrophobic interaction chromatography
  • cell culture supernatant or cell fractions containing IFN- ⁇ serve as starting material for the chromatographic purification in order to reach a sufficient purity which allows for its application for formulation of a pharmaceutical composition.
  • IFN- ⁇ intended for said purification is a polypeptide which exhibits biological and/or immunological features of naturally occurring human IFN- ⁇ and can be either a natural or a recombinant IFN- ⁇ .
  • glycosylated IFN- ⁇ more preferably recombinant IFN- ⁇ from eukaryotic host cells, preferably CHO cells, is used.
  • IFN- ⁇ species originated from the cell line BIC 8622 (ECACC 87 04 03 01) are used, which are for example described in European patent applications EP 287 075 and EP 529 300, the disclosures of which are referenced hereby.
  • the correct formation of the disulfide bond is essential for the biological activity.
  • the protein consists of 40% hydrophobic amino acids and is extremely hydrophobic (insoluble). Its pi is slightly basic (7.8-8.9).
  • the amino acid sequence shows four histidine residues in position 93, 97, 121 and 131, which explains the good binding to Me + * ligands.
  • the specific activity of IFN- ⁇ should be at least 2 x 10 8 IU/mg.
  • IFN- ⁇ preparations with high triantennary (>25%) and additional tetraantennary glycosylation (>5%) have been described, which can have a specific activity up to 3 x 10 IU/mg and more.
  • the two essential biological activities of IFN- ⁇ which can be measured are its antiviral and antiproliferative effect. Each of these biological activities can be measured by standard methods through inhibition of the cytopathic effect of a virus. A detailed description of the test methods used can be found in Stewart, W. E. 11 (1981), The Interferon System (Second, enlarged Edition), Springer- Verlag: Wien, New York; Grossberg, S.E.
  • the method of purifying IFN- ⁇ comprises two affinity chromatography steps, preferably performed before the hydrophobic interaction chromatography step.
  • the metal chelate affinity chromatography (Zinc Sepharose chromatography) yields are 90-100%, and the hydrophobic interaction chromatography (Butyl Sepharose chromatography) yields are > 70%, respectively.
  • Analysis by RP-HPLC as well as SDS-PAGE and subsequent silver staining confirmed that IFN- ⁇ was purified up to apparent homogeneity.
  • a band shift in SDS-PAGE under reduced and non-reduced conditions revealed that the internal disulfide bond was formed and thus the protein was correctly folded.
  • Isoelectric Focussing (IEF) Western blots the intermediate purification product and the final product exhibit a similar IFN- ⁇ isoform pattern as Avonex ⁇ .
  • the IFN- ⁇ preparation according to the present invention has been further analyzed via analytical SEC using Superdex 75 HR 10/30.
  • Major elution peaks at A280 and A214 exhibit a peak maximum between 13.9 and 14.0 ml elution volume.
  • the apparent molecular mass is 14 kDa, which indicates that the IFN- ⁇ monomers were eluted with a slight delay, probably due to an unspecific interaction with the column matrix.
  • the specific activity of the IFN- ⁇ purified according to the present invention already usually exhibits not less than 1 x 10 IU/mg, typically exceeding at least 2 x 10 IU/mg, and preferably exceeding at least 3 x 10 8 IU/mg and more.
  • the purification method for IFN- ⁇ comprises an anion exchange chromatography (AbXJ step, preferably directly applied after the HIC step in flow through modus. This further AEX step is especially advantageous for the preparation of pharmaceuticals composition of IFN- ⁇ , since in control experiments with additional spiking of samples with virus material, the obtained IFN- ⁇ preparation was no longer infectious and therefore is suitable for therapeutic application.
  • a cation exchange chromatography (CEX) step is omitted.
  • CEX cation exchange chromatography
  • CEX step is to be used as intermediary step and might be more suitable as a polishing step.
  • CEX cannot be directly applied after an affinity chromatography step with dye ligands such as Cibacron or metal chelate as IMAC because it requires increasing salt concentrations for elution. Therefore, subsequent rebuffering and desalting steps would become necessary.
  • dye ligands such as Cibacron or metal chelate as IMAC
  • the method of purifying IFN- ⁇ does not comprises the use of a preparative HPLC.
  • RP reverse phase
  • RP-HPLC would be applied, if required, only for analytical purpose.
  • immunoglobulin affinity chromatography is not part of said purification process of the present invention.
  • Those purification steps for therapeutic proteins are always associated with an extensive validation program in order to exclude security issues such as cross contamination. Therefore, omittance of said immunoaffinity steps is regarded as a big advantage.
  • the use of a hydroxyapatite chromatography is omitted.
  • the purification method according to the present invention makes only use of two to three different chromatography separation methods, in particular an affinity chromatography with dye ligands and/or metal chelates and a hydrophobic interaction chromatography, characterized by adsorption of the nonpolar surface regions of a protein at high salt concentrations to weak hydrophobic ligands in the stationary phase (salting effect) and elution by decreasing the buffer salt concentration.
  • this step is followed by an ion exchange chromatography step, based on the principle of a competitive interaction of charged ions, i.e. here anions.
  • the chromatographic purification of IFN- ⁇ comprises the following steps:
  • the IFN- ⁇ sample should be kept in a cationic environment; i.e., at pH values below its isoelectric point (pi), so that the used buffers and washing solutions, apart from some individual washing steps, preferably exhibit a pH of ⁇ 7; see also the Examples.
  • Blue Dextran Sepharose R or other suitable Cibacron R Blue immobilized matrices such as Matrex Gel Blue A from Amicon or Fraktogel 45 TSK AF-Blue from Merck or Blue-Sepharose R 6FF from GE Healthcare can be used.
  • suitable metal ions can be Cu 2+ , Zn 2+ , Co 2+ or Ni 2+ ions.
  • the desorption can be induced by competitive substances such as imidazol, histidine, glycine or NH 4 Cl, chelate agents as EDTA, IDA (iminodiacidic acid), TED (Tris-Carboxymethyl Ethylendiamine) or by lowering the pH value to pH 2 to 4.
  • Suitable separation media are immobilized iminodiacetic acid linked to agarose or to Fraktogel TSK HW-65F (Pierce) or Chelating Sepharose R FF (GE Healthcare) or Cellufine Chelate (Amicon).
  • the experiments performed according to the present invention revealed that particularly dye affinity chromatography with Cibacron Blue and IMAC chromatography, especially in combination, are particularly advantageous for the IFN- ⁇ purification process.
  • Cibacron in one preferred embodiment of the method of the present invention Cibacron
  • IFN- ⁇ is a strong binding partner of Cibacron Blue F3GA (CB-F3GA) and foreign bound proteins can be washed out by various buffers prior to elution. This strong interaction is most likely due to its enormous hydrophobicity. Since IFN- ⁇ quantatively binds even at low concentrations under physiological conditions, this step is especially suitable as a capture step, i.e., as a first chromatography step of the purification process in accordance with the present invention. Elution of IFN- ⁇ can be conducted for example with ethylenglycol, if necessary in a gradient. Blue Sepharose Streamliner or Blue Sepharose Fast Flow (GE)
  • a lectin affinity chromatography step can be considered, for example using Concanavalin A (ConA).
  • ConA Concanavalin A
  • the immobilized metal chelate chromatography (IMAC) is also often described for purification of IFN- ⁇ .
  • the carrier should be coupled with iminodiacetate (IDA). So far, IMAC was always performed after an affinity chromatography step (usually Cibacron Blue or ConA).
  • a hydrophobic interaction chromatography (HIC) is performed, wherein preferably butyl groups serving as ligands. So far, the use of hydrophobic interaction chromatography for the purification of IFN- ⁇ has not been investigated in detail. Due to the extreme hydrophobicity of IFN- ⁇ , adsorption to and desorption from the hydrophobic matrix seemed to be problematical. In experiments conducted in accordance with the present invention, it was surprisingly found that IFN- ⁇ bound and easily eluted in the HIC step, especially when an acetate buffer with pH 5.0 is used for application and elution steps.
  • HIC can be used as a capture step (requires addition of salt prior to applying the sample) and an intermediate chromatography step after the metal chelate chromatography such as IMAC and, if appropriate, can be performed directly after the dye affinity chromatography, e.g., with Cibacron.
  • IMAC metal chelate chromatography
  • early inactivation of potentially present enzymes or viruses can be achieved in the HIC by elution with organic solvents.
  • HIC HIC
  • strongly hydrophobic proteins are short-chained alkyls like methyl, butyl or propyl, for example Butyl Sepharose 4 Fast Flow, Macro Prep Methyl, Fractogel EMD Propyl or Phenyl Sepharose Low Substitution (Merck). Since also the matrix contributes to binding, it had to be tested in the experiments according to the present invention, which material is finally best suited for the purification of IFN- ⁇ . In this context it turned out that butyl groups are most appropriate for absorption and in particular subsequent desorption; see also the Examples. Products of Amersham Biosciences (now GE Healthcare) can be used.
  • a membrane with quaternary amino groups is used for anion exchange membrane filtration.
  • the person skilled in the art can obtain product information on suitable matrices and protocols for performing the anion exchange chromatography from the supplier such as Amersham Biosciences (http://www.amershambiosciences.com, now GE Healthcare) or Bio-Rad (http://www.bio- rad.com).
  • 20 mM sodium acetate pH 5.0 is used for equilibration and washing in the anion exchange chromatography step.
  • Further suitable conditions for anion exchange chromatography can be found in the literature like in the handbook "Ion Exchange Chromatography - Principles and Methods" from Amersham Biosciences, Freiburg, Kunststoff (now GE Healthcare), 2002.
  • the purification process for IFN- ⁇ comprises therefore at least one of the follwoing steps; (e) an ultrafiltration (UF) step;
  • ultra- and microfiltration serve for the specific purification and concentration of IFN- ⁇ while size exclusion chromatography and nanofiltration are especially used for the removal of host cell DNA, endotoxins and remaining process related impurities of the eluates, if present.
  • ultrafiltration is a tangential flow filtration with a size exclusion of 5 kD - 1000 kD, and for microfiltration a 0.2 ⁇ m membrane, for the size exclusion chromatography Superdex 200 and/or for the nanofiltration a filter with a pore size of 15 - 75 nm should be used.
  • the claimed purification process for IFN- ⁇ comprises the following steps as illustrated in the Examples: ⁇ a; a aye aiiimty chromatography(AC) step;
  • the present invention also relates to a pharmaceutical composition, comprising the IFN- ⁇ obtained in accordance with the method of the present invention.
  • IFN- ⁇ obtained can be stored as lyophilisate or preferably in liquid form. It can be applied subcutaneous or intravenous.
  • Suitable pharmaceutically acceptable carrier for the formulation of recombinantly expressed IFN- ⁇ are stabilizers like sugar or sugar alcohols, amino acids as well as tensides like Polyssorbate 20 or 80 as well as suitable buffer substances. Examples for formulations are described in international application WO98/28007 and WO99/15193 as well as in European patent application EP 0 529 300, see also products of Avonex® and Rebif® in ROTE LISTE 2005.
  • the present invention also relates to a method for the preparation of a pharmaceutical liquid formulation of human IFN- ⁇ suitable for parenteral application comprising a method for the purification of IFN- ⁇ as described herein before and in the Examples, and
  • the IFN- ⁇ formulation can be stored for example in suitable washed and sterilized glass vials (hydrolytic class 1) with a pharmaceutical acceptable rubber plug.
  • the pnarmaceuticai ifN- ⁇ formulation can also be filled into antiseptic pre-packaged syringes or in capsules or carpules for self injection devices and used for self injection.
  • the aqueous solution can be freeze-dried - although this is not preferred - by addition of further additional carriers known by the person skilled in the art and is available in liquid form after reconstitution.
  • suitable preservatives liquid multiple dosage forms can be produced as well as ophthalmic solutions and drop solutions for oral application.
  • the pharmaceutical composition of IFN- ⁇ comprises acetate, NaCl or one of the amino acids arginine, lysine und glutamine either alone or in addition to one or more further carriers, wherein the carrier is preferably methionine, mannitol, sorbitol, glycerol or a tenside, wherein the tenside is preferably Polysorbate 20 or 80.
  • the pH value of the formulation preferably ranges between 4.3 and 4.8.
  • the specific activity of the purified IFN- ⁇ according to the present invention is usually at least 1 x 10 8 IU/mg, typically at least 2 x 10 8 IU/mg, preferably at least 3 x 10 8 IU/mg and more.
  • a multitude of possible buffer compositions has been identified as suitable for formulation, providing apparently homogenous IFN- ⁇ purified by a dye affinity chromatography (AC), metal chelate affinity chromatography (MAC) and hydrophobic interaction chromatography, which is substantially stable and biologically active at room temperature as well as at -80°C storage and after subsequent thawing.
  • the stability of IFN- ⁇ in a given buffer at a concentration of 200 ⁇ g/ml at storage up to two weeks at +4°C or at - 80°C amounts to at least 95%, preferably at least 97% and possibly nearly 100% of the initial activity. Therefore, the present invention also particularly relates to a pharmaceutical composition comprising biologically active IFN- ⁇ .
  • IFN- ⁇ preparations are preferably stable over a storage time of 26 to 27 days at -80 °C and of 47 to 48 days (> 6.5 weeks) at +4 0 C, as confirmed by RP-HPLC measurements, A280 measurement and/or the determination of the A320 value ("turbidity" below 0.010).
  • the IFN- ⁇ isoform pattern in Western blots after SDS-PAGE and on IEF Western blots is preferably very similar or identical to Avonex ⁇ and to the IFN- ⁇ preparation after the HIC purification step.
  • the IFN- ⁇ preparations according to the present invention preferably exhibit an apparent molecular mass of 12-16 kDa.
  • the buffer designated 3a of table 10 is particularly well suited for storage of recombinantly produced glycolysated IFN- ⁇ .
  • the present invention relates to a pharmaceutical composition, comprising IFN- ⁇ in 25 mM acetate, 150 mM NaCl und 0.167 % (v/v) Polysorbate 20 and which preferably has a pH value of pH 4.8.
  • the liquid pharmaceutical formulations according to the present invention are preferably substantially free of human serum albumin and more preferably— apart from the pharmaceutical agent - free of human or animal polypeptides, in particular of serum proteins.
  • the stability of the IFN- ⁇ formulation can further be positively affected by sparging with an inert gas such as helium or nitrogen. This is particularly true for the present IFN- ⁇ formulation in a suitable receptacle or container, wherein the head space of said receptacle or container is preferably also sparged with an inert gas such helium or nitrogen, and preferably wherein the head space is not exceeding 30% of the volume of the receptacle or container.
  • an inert gas such as helium or nitrogen
  • the present invention also relates to a medicament, comprising purified IFN- ⁇ obtained by the method of the present invention and pharmaceutical acceptable carriers as buffer, salts, tensides and stabilizers.
  • the liquid formulation of IFN- ⁇ is stable over a long time period and can basically be stored in any suitable receptacle or container.
  • the present invention also relates to a receptacle or container comprising a liquid pharmaceutical formulation of human IFN- ⁇ suitable for parenteral application and obtainable by method of the present invention for the preparation of the pharmaceutical liquid formulation of IFN- ⁇ as described hereinbefore and in particular in the Examples.
  • the receptacle or container according to the present invention is preferably such that its inner surfaces which are in contact with the pharmaceutical formulation prevent the adsorption of IFN- ⁇ .
  • at least one surface of the receptacle, or container which is in contact with the liquid formulation is coated with a material or composed of a material essentially consisting or made of polypropylene (PP), silicone or polytetrafluorethylene or ethylene tetrafluorethylene (ETFE) copolymer.
  • PP polypropylene
  • ETFE ethylene tetrafluorethylene
  • the receptacle is a container that is conventionally intended for the storage and/or administration of a liquid medicament like a vial, syringe, ampoule, carpule, puncture bottle or infusion container, wherein the liquid formulation of IFN- ⁇ according to the present invention is particularly advantageous for the use in pre-filled syringes or ampoules.
  • the liquid formulation is present for example in a syringe or an ampoule at a concentration of IFN- ⁇ of 10 - 500 ⁇ g/ml, preferably 50 - 250 ⁇ g/ml and/or an activity of 5 - 50 million I.E./ml.
  • compositions of the present invention obtainedas well as the receptacles and containers containing these compositions can be used for the treatment of tumors, virus diseases, immunopathies or inflammations including rheumatic diseases, allergies, psoriasis, Crohn's disease and degenerative diseases of the nervous system, in particular multiple sclerosis.
  • the required quantity of recombinant IFN- ⁇ in a medicament for the desired therapeutic effect depends on the respective administration and treated subject as well as the respective disease.
  • a suitable dosage of the active integrient for administration on a human is ranging between 0.1 x 10 6 and 100 x 10 6 I.E.
  • compositions according to the present invention and the receptacles and containers containing them are preferably designed for ophthalmological, subcutan, intracutan, intramuscular, intravenious, intrathecal, intraarticular, intratumoral/peritumoral, intralesional/perilesional or topic application.
  • the IFN- ⁇ liquid medicament according to the present invention can be used for immediate administration, for example in a kit.
  • the present invention therefore also relates to a kit for the administration of IFN- ⁇ by infusion or injection, comprising one or more of the above described receptacles, preferably along with instructions for storage and/or administration.
  • IFN- ⁇ administration at a dosage of 1 x 10 6 to 1O x 10 6 I.E. will be provided, wherein also lower or higher dosages may be indicated, however, depending on the medical indication and stage of disease.
  • several receptacles are provided in the kit according to the present invention, for example for weekly intramuscular administration for one month 4 pre- filled syringes with needles or 4 puncture bottles along with pre-packed syringes and needles as well as, if appropriate, a solvent in case lyophilisate is used which is basically possible, but however not preferred.
  • Rebif ⁇ is intraveniously administrated three times a week.
  • the kit according to the present invention advantageously has safety compartments for syringes, injections and/or infusions needles, respectively.
  • discharge aids for the needles and prepared or pre-fitted sealing caps are also to be considered.
  • the liquid IFN- ⁇ formulations according to the present invention are stable over a long time period, in particular at about 2-8°C, preferably over a period of at least 4 weeks. Therefore, the liquid formulations, receptacles and kits according to the present invention can advantageously be stored in a conventional refrigerator.
  • IFN- ⁇ as a starting point typically is human recombinant IFN- ⁇ Ia expressed by recombinant Chinese Hamster Ovary (CHO) cell line.
  • the protein contains arginine N-linked glycanes, exhibiting an isoform profile as described in the relevant draft Ph.Eur. monograph (PHARMEUROPA) Vol. 15, Nr. 4, October 2003.
  • the specific activity is demonstrated in the CPE assay (CPE, cytopathic effect) by use of A549 cells and enzephalomyocarditis virus EMC as infections agent (PHARMEUROPA, Vol. 15, Nr. 4, October 2003).
  • the specific activity of purified bulk IFN- ⁇ is approximately 3.2 x 10 IU x mg "1 .
  • a recombinant CHO cell line is generated, which expresses human IFN- ⁇ Ia, and is adapted to suspension and serum-free culture conditions.
  • expression vector a vector can be used which contains the (natural) human IFN- ⁇ gene sequence, a translation start sequence according to Kozak (Kozak sequence) and as a regulatory element for expression the SV40 promoter with a SV40 polyA terminator sequence. The selection and amplification of the expression vectors take place by a murine dhfr gene sequence under the control of an Adeno major late promotor and the SV40 polyA sequence.
  • the parental cell line is preferably a dhfr- deficient Chinese Hamster Ovary (CHO) cell line obtainable for example from ATCC or DSMZ.
  • the generation of the producer cell line is made in accordance with methods known in the art.
  • the dhfr deficient CHO cells are transfected with the expression vector. After selection, subcloning utilizing cloning cylinders and subsequent amplification using methotrexate, the resulting cell line is adapted to serum free culture condition and is tested as suspension culture. A second round of subcloning can be performed utilizing the limited dilution technique.
  • the cell culture process consists preferably of seed train expansion of transfected CHO cells in Erlenmeyer flasks followed by commercial scale production in a 10 liter bioreactor.
  • the purification process comprises several steps designed to yield a product with high biological activity and biochemical characteristics, and which at the level of product - and product - related substances and impurities are in full compliance with the current regulatory, scientific and compendia standards.
  • the scope for this development phase includes an extensive optimization of each purification step and to generate the representative material for a pilot PK/PD-study with Cynomolgus monkeys. Due to limited development resources, the purification (excluding capture step) of this material is performed in a scaled down laboratory process. a) Affinity chromatography: Streamline Blue Sepharose
  • a washing buffer is used a): 20 mM NaH 2 P(VNa 2 HPO 4 , IM NaCl, pH 7.2.
  • Column regeneration is performed with regeneration buffer 1 : 50 mM Tris/HCl, 1 M NaCl, pH 7.6;
  • regeneration buffer 2 10 mM Tris-HCl, 800 mM NaCl, 50 mM EDTA, 10 % Isopropanol, pH 7.6
  • regeneration buffer 3 50 mM Tris/HCl, 3 M NaCl, pH 7.6 together with SIP: 70% Ethanol > 12h and 2h/CIP: 0.5 M NaOH.
  • the column is stored in storage buffer: 0.01 M NaOH preferably at 2-8°C.
  • Conductivity of the eluate is determined and adjusted to 2.5 ⁇ 0.5 mS/cm using 20 mM Na- acetate, pH 5.0 (approximately dilution factor T). The pH is adjusted to pH 5.0 ⁇ 0.1.
  • the column is equilibrated with washing buffer a) and after elution of the samples treated with SIP/CIP: 0.1 M NaOH and is stored in storage buffer: 20 mM NaOH. d) Anion exchange membrane filtration
  • Loading buffer used for equilibration is sparged with nitrogen in order to remove oxygen prior to equilibration, and after subsequent elution of the sample the column is treated with IM NaOH and is stored in storage buffer: 20 mM NaOH. f) pre-filtration / Nanofiltration
  • the purified material is filtered prior to nanofiltration according to table 8.
  • Mini Kleenpak (Pall Systems) is used during filtration in order to protect the nanofiltration membrane for micro-precipitates .
  • the purified material is filtered through a Planova N20 filter device (table 9).
  • a Planova N20 filter device for virus filtration the hollow fiber system of Planova 2ON (Asahi Kasei) is appropriate regarding the yield to IFN- ⁇ and the compatibility with a use drug substance storage buffer. This method yields in 90 to 100%. The purity was > 99% (determined by RP- HPLC).
  • the nanofilter is washed with up to 300 mL 2OmM NaAc, pH 5.0, 150 mM NaCl, 0.167% Tween 20 (v/v). Thereafter, the purified drug substance is filled into containers for freezing and storage (TPP Cryotubes).
  • the total yield of IFN- ⁇ in relation to the starting activity is 25%.
  • Example 3 Identification of a suitable storage buffer system for IFN- ⁇ drug substance
  • IFN- ⁇ Blue Sepharose eluates are purified by zinc Sepharose chromatography and butyl Sepharose chromatography and are concentrated to approximately 1.9 mg/ml using Vivacell 70 centrifugal filter device.
  • This preparation is the starting material needed for the formulation studies.
  • the buffer exchange is performed using Size Exclusion Chromatography (NAPlO, GE Healthcare). A total of 20 different buffers are investigated (see table 10), partly sparged with nitrogen as inert gas. Furthermore, the influence of the head space and closure system on storage stability and precipitation is analyzed.
  • Non-oxidizing conditions the buffers are 0.2 ⁇ m filtered using vacuum filtration and sparged with N 2 for 10 min before use; after filing of the formulated IFN- ⁇ into the PP cryovials the head space (maximally 30 % of the cryovial volume) is filled with 0.2 ⁇ m filtered N 2 .
  • Oxidizing conditions Buffers not 0.2 ⁇ m are filtered using vacuum filtration, but are sparged with air for 10 min before use; after filling of the formulated IFN- ⁇ into the PP cryovials the head space are not filled with N 2 .
  • Non-oxidizing/semi-sterile conditions The buffers are first sparged with N 2 for 30 min, then sterile filtered through a 0.2 ⁇ m cellulose acetate membrane (Schleicher & Schuell; FP 30/02 CA-S) under a lamina flow; the following work are performed under a lamina flow; the final protein solution are not sterile filtered because a significant loss of IFN- ⁇ is expected; after filling of the formulated IFN- ⁇ into the PP cryovials the head space (maximally 30% of the cryovial volume) is filled with 0.2 ⁇ m filtered N 2 .
  • IFN- ⁇ protein stability measured by the means of RP-HPLC measurement, showing good stability for all buffer eluates after storage for 12 to 14 days at +4°C (97-103% recovery) with the exception of buffer HaI which exhibit a lower IFN- ⁇ concentration (approximately
  • the A320 values is especially low (0.000-0.006) for buffer eluates 3a (25 mM acetate, 150 mM NaCl, 0.167% Tween 20, pH 4.8), 9al (25 mM acetate, 150 mM NaCl, 25 % (v/v) glycerol, pH 3.0) and 9bl (25 mM acetate, 150 mM NaCl, 25 % (v/v) PEG300, pH 3.0) before the storage and after storage at both +4 0 C and -80 0 C, and for two more buffer eluates containing low IFN- ⁇ concentration (approximately 60 ⁇ g/ml) before the storage and after storage at +4 0 C.
  • the most suitable buffer for the drug substance with respect to a storage of IFN- ⁇ at -80°C (a demand of the preliminary monograph for IFN- ⁇ ) regarding the yield after thawing and the absence of precipitation is used for pilot and commercial scale (25 mM NaAc, pH 4.8, 150 mM NaCl, 0.167% Tween 20).

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Abstract

L'invention porte sur un procédé de production d'interféron-bêta (IFN-β) glycosylé humain, comprenant deux étapes de chromatographie d'affinité suivies par une étape de chromatographie par interaction hydrophobe, de préférence avec une étape subséquente de chromatographie d'échange d'anions. L'IFN-β obtenu par le procédé tel que décrit est caractérisé par une pureté élevée et par une activité biologique spécifique élevée, en raison du fait qu'il est particulièrement approprié pour la production de compositions pharmaceutiques.
PCT/EP2010/004130 2009-07-07 2010-07-07 Procédé de purification d'interféron-bêta WO2011003600A1 (fr)

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US13/382,807 US20120177603A1 (en) 2009-07-07 2010-07-07 Method for the purification of interferon-b
EP10737494A EP2451470A1 (fr) 2009-07-07 2010-07-07 Procédé de purification d'interféron-bêta
JP2012518819A JP2012532167A (ja) 2009-07-07 2010-07-07 インターフェロンβの精製方法
EA201290040A EA201290040A1 (ru) 2009-07-07 2010-07-07 СПОСОБ ОЧИСТКИ ИНТЕРФЕРОНА-β

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DE102009032179.9 2009-07-07
DE102009032179A DE102009032179A1 (de) 2009-07-07 2009-07-07 Verfahren zur Reinigung von Interferon beta

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