EP2283036A1 - Procédé de purification d'érytropoïétine - Google Patents

Procédé de purification d'érytropoïétine

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Publication number
EP2283036A1
EP2283036A1 EP09757442A EP09757442A EP2283036A1 EP 2283036 A1 EP2283036 A1 EP 2283036A1 EP 09757442 A EP09757442 A EP 09757442A EP 09757442 A EP09757442 A EP 09757442A EP 2283036 A1 EP2283036 A1 EP 2283036A1
Authority
EP
European Patent Office
Prior art keywords
chromatography
erythropoietin
anion exchange
exchange chromatography
hydroxyapatite
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.)
Withdrawn
Application number
EP09757442A
Other languages
German (de)
English (en)
Inventor
Wolfgang Wienand
Franz-Rudolf Kunz
Dietmar Reichert
Wilfried Eul
Rudolf Hanko
Christian Birr
Monika Singhofer-Wowra
Dagmar SCHOPOHL-KÖNIG
Lars Faber
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.)
Evonik Operations GmbH
Original Assignee
Evonik Degussa GmbH
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
Application filed by Evonik Degussa GmbH filed Critical Evonik Degussa GmbH
Publication of EP2283036A1 publication Critical patent/EP2283036A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]

Definitions

  • the present invention relates to a method for the purification of erythropoietin, wherein erythropoietin-containing culture supernatant of erythropoietin-producing eukaryotic cells is processed by certain chromatographic purification steps carried out in a fixed manner one after the other.
  • EPO Erythropoietin
  • EPO Erythropoietin
  • EPO is primarily produced in the kidneys and from there via the bloodstream to its destination.
  • the damaged kidneys produce too little or no EPO at all, with the result that too few erythrocytes emerge from the stem cells of the bone marrow.
  • This so-called renal anemia can be treated by administering EPO in physiological amounts that stimulate the formation of erythrocytes in the bone marrow.
  • the EPO used for administration can either be obtained from human urine or generated by genetic engineering methods. Since EPO is present in trace amounts in the human body, isolating EPO from the natural source is virtually impossible for therapeutic applications. Therefore, genetic engineering methods provide the only economic opportunity to produce this substance in larger quantities.
  • erythropoietin The recombinant production of erythropoietin is usually carried out in CHO host cells. While these were formerly cultivated in culture media to which fetal calf serum and sometimes also bovine insulin had been added, cultivation nowadays regularly takes place in serum- and protein-free medium. In this way, the risk of contamination with bovine proteins, bovine viruses, bovine DNA or other unwanted substances of bovine origin can be completely avoided.
  • suitable serum and protein free media are available from various commercial manufacturers, e.g. the medium MAM-PF2, marketed by, among others by Bioconcept, Allschwil, Switzerland, or the media DMEM and DMENU12, offered e.g. by Invitrogen / Gibco, Eggenstein, Germany.
  • EP-A-0 228 452 describes a process for purifying biologically active erythropoietin from a liquid, comprising the chromatographic steps of anion exchange chromatography and reversed-phase chromatography.
  • EP-A-0 267 678 describes the purification of an erythropoietin prepared in serum-free culture, followed by dialysis, ion exchange chromatography, preparative reversed-phase HPLC and gel filtration chromatography.
  • the gel filtration chromatography step can be replaced by ion exchange chromatography. It is also proposed to carry out dye affinity chromatography on a blue trisacryl column before (first) ion exchange chromatography.
  • EP-AO 830 376 describes a process for the purification of erythropoietin, in which EPO from the culture supernatant is subjected to dye affinity chromatography in the first step of the chromatographic purification. The second step is followed by chromatography on a hydrophobic support, followed by hydroxyapatite chromatography. This is followed by reversed-phase HPLC, followed by anion exchange chromatography as the last chromatography step.
  • EP-A-1 127 063 describes a purification process for
  • Erythropoietin comprising the following steps: differential precipitation, hydrophobic interaction chromatography, diafiltration, anion exchange chromatography, cation exchange chromatography and size exclusion chromatography.
  • the individual purification steps are carried out in EP-A-1 127 063 in the order mentioned.
  • purification comprises the following steps: differential precipitation, hydrophobic interaction chromatography, diafiltration, anion exchange chromatography, cation exchange chromatography, further diafiltration and size exclusion chromatography.
  • the method provides for precipitation followed by centrifugation.
  • gel filtration is mandatory to complete the chromatographic purification.
  • WO2005 / 121173 specifies a process for the purification of EPO produced by means of fermentative processes. This method is based on a chromatographic purification with at least four different chromatographic separation methods. The first anion exchange chromatography is followed by affinity chromatography, hydrophobic interaction chromatography and hydroxyapatite chromatography, the order of these 3 latter types of chromatography being arbitrary. Finally, anion exchange chromatography is again used.
  • EPO which satisfies the purity criteria required by the European Pharmacopoeia.
  • Criteria for a suitable EPO include i.a. a content of foreign proteins originating from the host cell of ⁇ 100 ppm, content of DNA from the host cell of ⁇ 100 pg / mg EPO and finally a composition which corresponds to the standard with respect to the isoform composition (Ph. Eur., 01/2002: 1316).
  • Object of the present invention is to provide a further purification process.
  • This should provide an EPO end product that complies with the European Pharmacopoeia (Ph. Eur. 01/2002: 1316) standard or the Guidance on Biosimilar Medicinal Products Containing Recombinant Erythropoietins (EMEA / CHMP / 94256/2005), and in a suitable manner in a technical, especially fermentative EPO production is used.
  • the present method should, from the economic point of view, be superior to the state of the art.
  • the process according to the invention should not require any significant worsening of the cleaning performance with fewer chromatographic purification steps and should be able to do without special technically complicated chromatography steps, such as, for example, affinity chromatography.
  • the technical object is achieved by a method for the purification of erythropoietin, wherein erythropoietin-containing culture supernatant of erythropoietin-producing eukaryotic cells is subjected to the following steps: a) removal of cell constituents; and b) treating the product from a) by the following Chroma ⁇ tographie suitse in the order given i) reversed-phase chromatography; ii) anion exchange chromatography; iii) hydroxyapatite chromatography.
  • step a) In a preferred embodiment of the method of any ⁇ their chromatographic methods are applied before step a) and between the steps a) to b iii). Moreover, it is further preferred that) no other Chro ⁇ chromatography methods are also applied to step b iii.
  • an erythropoietin product is produced that the poeia in of the European Pharmacopeia (Ph.Eur .; 01/2002: 1316) standard defined or guidance on biosimilar medicinal products Containing Recombi ⁇ nant erythropoietin (EMEA / CHMP / 94256/2005).
  • the erythropoietin so produced meets the following Kri ⁇ criteria: a content of foreign proteins from the host cell derived from ⁇ 100 ppm, content of DNA from the host cell of ⁇ 100 pg / mg EPO and finally in terms of isoforms composition standard corresponding Caribbeanset ⁇ injection (Ph. Eur .; 01/2002: 1316).
  • the resulting EPO product also has a biological activity of at least 150,000 IE / mg in bioassay.
  • the band structure in the IEF and the glycosylation pattern correspond to a commercial Erypo® preparation.
  • chromatographic principles used in the process according to the invention are known from the literature and are available to the technical man (Meyer, practice of high-performance liquid chromatography, Wiley-VCH Weinheim 2004, Unger, Handbook of HPLC, Part 1 and 2, GIT Verlag Darmstadt 1994). In addition, detailed and detailed information on the chromatography media can be found in the product information of the respective manufacturer or supplier.
  • the eukaryotic erythropoietin-producing cells mammalian cells, more preferably human cells, and most preferably Chinese hamster ovary cells (CHO), express human recombinant erythropoietin.
  • an ultrafiltration is carried out after steps i), ii) and / or iii).
  • step a) comprises microfiltration and subsequent ultrafiltration.
  • the reversed-phase chromatography is carried out on a carrier material as a stationary phase, which is selected from the group Ci to Cs-modified silica gels, hydrophobized polymer supports based on polystyrene / divinylbenzene and hydrophobized monolithic phase materials on silica gel. or polymer base, and an aqueous alkanol solution is used as eluent.
  • a carrier material is selected from the group Ci to Cs-modified silica gels, hydrophobized polymer supports based on polystyrene / divinylbenzene and hydrophobized monolithic phase materials on silica gel. or polymer base, and an aqueous alkanol solution is used as eluent.
  • a carrier material as a stationary phase
  • a carrier material as a stationary phase which is selected from the group Ci to Cs-modified silica gels, hydrophobized polymer supports based on polystyrene /
  • the anion exchange chromatography is carried out on a support material as a stationary phase having functional groups which are selected from diethylaminoethyl groups (DEAE), quaternary aminoethyl groups (QAE), quaternary ammonium groups or dimethylamines. noethyl groups (DMAE).
  • DEAE diethylaminoethyl groups
  • QAE quaternary aminoethyl groups
  • DMAE noethyl groups
  • the anion exchange chromatography comprises at least one washing step with an aqueous buffer system, preferably with a buffer system based on an organic acid, in particular with an acetate buffer.
  • the anion exchange chromatography comprises at least one washing step with an aqueous buffer solution having a pH of 3.5 to 5.5, more preferably having a pH of 4.0 to 5.0, and most preferably having a pH of about 4 ; 5.
  • an aqueous buffer solution having a pH of 3.5 to 5.5, more preferably having a pH of 4.0 to 5.0, and most preferably having a pH of about 4 ; 5.
  • Particularly suitable as a buffer is an acetate buffer, preferably a sodium acetate buffer.
  • the eluent used in the anion exchange chromatography is preferably inorganic acid in an aqueous buffer solution.
  • chloride ions in an aqueous buffer solution are used as the eluent in anion exchange chromatography.
  • the hydroxyapatite chromatography comprises at least one washing step with an organic acid-based buffer system, preferably with an acetate buffer.
  • an organic acid-based buffer system preferably with an acetate buffer.
  • a buffer system based on an inorganic acid particularly preferably a phosphate buffer.
  • the present invention provides a method of purifying erythropoietin, wherein erythropoietin-containing culture supernatant of erythropoietin-producing eukaryotic cells is subjected to the steps of: a) removing cell constituents; and b) treating the product from a) by the following chromatographic steps in the order given: i) reversed-phase chromatography; ii) anion-exchange chromatography; iü) hydroxyapatite chromatography.
  • Reversed-phase chromatography is used as a "capture step.”
  • EPO is enriched from the fermentation supernatant, whereby different hydrophobic interactions between the sample molecules and the stationary phase are important.
  • Preferred reversed-phase materials are nonporous polystyrene / divinylbenzene-based hydrophobic polymer supports. Particularly preferred are SOURCE 30RPC or the corresponding 15 ⁇ m material (SOURCE 15RPC) from GE Healthcare.
  • the eluents used can preferably be alcohols such as ethanol or 2-propanol or mixtures thereof with appropriately buffered aqueous systems. Particularly preferred is the use of 2-propanol, since the organic solvent content in the eluent, compared to ethanol, is significantly lower and so there are safety and economic advantages. Moreover, due to its miscibility and solubility properties, 2-propanol is particularly suitable as a solution mediator (Unger, Handbuch der
  • the anion exchange chromatography is based on the competitive interaction of charged ions of the sample charge with the buffer medium used. It can be carried out with conventional, commercially available anion exchange resins with diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), quaternary ammonium or dimethylaminoethyl (DMAE). These phase materials are available, for example, from GE Healthcare, Tosoh Biosience, Bio-Rad or Merck. Preference is given to using diethylaminoethyl (DEAE) -functionalized anion exchange resins. TSKgel DEAE-5PW (30 ⁇ m), available from Tosoh Bioscience, is particularly preferably used in the anion exchange chromatography.
  • the anion exchange chromatography comprises an acidic wash, with which the basic isoforms of erythropoietin are eluted by the pH reduction and thus separated off (EP-1428878) the pH of the wash buffer is between 3.5 and 5.5, more preferably between 4.0 and 5.0, and most preferably about 4.5.
  • a buffer especially a sodium acetate buffer is suitable.
  • hydroxyapatite chromatography conventional hydroxyapatite (or hydroxyapatite) materials can be used. Hydroxylapatite is hexagonal crystalline calcium phosphate and is particularly suitable for the separation of proteins and other biopolymers. This purification step is carried out to remove the "phosphated" EPO molecules, preferably CHT ceramic hydroxyapatite (Bio-Rad), more preferably CHT ceramic hydroxyapatite type 1 (Bio-Rad).
  • CHT ceramic hydroxyapatite Bio-Rad
  • CHT ceramic hydroxyapatite type 1 Bio-Rad
  • Both reversed-phase chromatography and anion exchange chromatography, as well as hydroxyapatite chromatography, can be repeated in order to increase the overall product yield in terms of reprocessing for those eluate fractions which do not have the product quality, in particular their band or glycosylation pattern Correspond to reference material. These are typically the early or late edge fractions of the main fractions, which as such already have the desired product properties after the first purification step.
  • the grading of the fractions of the various purification steps is carried out by standard methods (isoelectric focusing (IEF) and high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD): Lasne, Nature 2000, 405 605-635, Hollander et al , LaborPraxis 2004, 56-59; Hokke et al., Eur. J. Biochem. 1995, 228, 981-1008; Dionex Technical Note 42, 1997.
  • the reference material used in each case is a commercially available Erypo® preparation (JANSSEN-CILAG).
  • microfiltration is carried out via 1.2 ⁇ m and 0.65 ⁇ m filters for cell separation. Subsequently, an ultrafiltration (cut off 10,000 Da) of the cell-free filtrate to 1/10 of the starting volume. After ultrafiltration, the concentrated cell supernatant is sterile filtered and used in the first chromatographic step (reversed-phase chromatography).
  • filtration steps in particular the sterile filtration, are known from the literature and familiar to the person skilled in the art (Munir, Handbook Ultrafiltration, Behr Hamburg 1990, Ullmann B2, 10-2, 10-21 and B3 11-6, Gasper, Handbook of Industrial Solid-Flow Filtration, Huthig Heidelberg 1990, Ullmann Al 6, 187-258).
  • the EPO obtained by the process according to the invention fulfills the quality criteria defined in the European Pharmacopoeia.
  • the isoform composition and the glycosylation pattern correspond to the standard defined in the European Pharmacopoeia (Ph.Eur.E01 / 2002: 1316) or the Guidance on Biosimilar Medicinal Products Containing Recombinant Erythropoietins (EMEA / CHMP / 94256/2005).
  • the reference material used is a commercially available Erypo® preparation (JANSSEN-CILAG).
  • the activity of the protein should be at least 100,000 IU / mg, preferably at least 125,000 IU / mg and more preferably at least 150,000 IU / mg (see also Europaische Pharmacopoeia 01/2002: 1316).
  • the EPO purified according to the invention is preferably recombinant human erythropoietin produced in eukaryotic cells.
  • the recombinant EPO is in mammalian cells, particularly preferably produced in CHO cells, as described, for example, in EP-AO 205 564 and EP-AO 148 605.
  • the fermentation is carried out according to conventional protocols in commercially available culture media.
  • erythropoietin is understood as meaning any protein which is capable of stimulating erythrocyte formation in the bone marrow and according to the assay described in the European Pharmacopoeia (Ph. Eur., 01/2002: 1316) can be clearly identified as erythropoietin (determination of activity in polycythemic or normocytamic mice).
  • the erythropoietin may be the wild-type human erythropoietin or a variant thereof having one or more amino acid substitutions, deletions or additions.
  • this variant is replaced only by 1 to 20, preferably only 1 to 15, more preferably in only 1 to 10 amino acid positions of the human wild-type erythropoietin by amino acid substitutions, deletions or additions.
  • Purification of erythropoietin or enrichment of erythropoietin means in the present case that the protein erythropoietin is obtained from a mixture in a very pure form, that is, the erythropoietin contained in the mixture is enriched until substantially no further proteins are present in addition to standard rythropoietin are included.
  • FIG. 1 shows, by way of example, the chromatographic separation of the erythropoietin peak in reversed-phase chromatography. Plotted is the peak intensity in the form of milli-absorption units (mAU) over the elution time (in min.) At a UV wavelength of 280 nm.
  • FIG. 2 shows, by way of example, the chromatographic separation of the erythropoietin peak under the conditions of anion exchange chromatography. The peak intensity is plotted as milli-absorption units (mAU) over the elution time (in min.) At a UV wavelength of 280 nm.
  • mAU milli-absorption units
  • FIG. 3 shows by way of example the IEF gel of the isolated EPO eluate fraction after anion exchange chromatography in comparison to the Erypo® preparation.
  • FIG. 4 shows by way of example the chromatographic separation of the erythropoietin peak in the case of the hydroxyapatite
  • FIG. 5 shows, by way of example, the IEF gel of the isolated EPO eluate fraction after hydroxyapatite chromatography in comparison to the Erypo® preparation.
  • Figure 6 shows the native glycosylation status of the purified final EPO product.
  • FIG. 7 shows the glycosylation status of a commercially available Erypo® preparation (JANSSEN-CILAG).
  • EPO is produced fermentatively in CHO cells.
  • the fermentation is carried out according to standard methods, as described in the patent and scientific literature for eukaryotic, in particular CHO cells.
  • the cultivation takes place in the perfusion reactor in culture medium that is free from animal components.
  • the harvest takes place continuously over a period of up to 50 days.
  • microfiltration is performed using a suitable filter (eg Opticap XLT30 Capsule with Milligard Medium 0.5-l, 2 ⁇ m, Millipore and Sarotbran P Midi-caps 0.45 - 0.65 ⁇ m, Sartorius) Flow rate of 1-2 L / min performed.
  • a suitable filter eg Opticap XLT30 Capsule with Milligard Medium 0.5-l, 2 ⁇ m, Millipore and Sarotbran P Midi-caps 0.45 - 0.65 ⁇ m, Sartorius
  • an ultrafiltration (cut off 10,000 Da) of the cell-free filtrate eg Ultran Pilot, polyethersulfone 4 ⁇ 0.45 m 2 , Schleicher & Schull
  • a sterile filtration eg via Opticap filter units with 0.5 ⁇ m Milligard Pre-filter and 0.2 ⁇ m Durapore sterile filter, Millipore.
  • Reversal phase chromatography on SOURCE 30RPC is carried out as a "capture step.”
  • EPO is enriched from the fermentation supernatant
  • several washing steps are carried out, and the first washing step is carried out with PBS buffer (phosphate buffered saline)
  • the next washing step is carried out with a mixture of water / isopropanol / TFA in a volume ratio of 10/2 / 0.1 to 10/1 / 0.1 at a flow rate of 40-50 mL / min.
  • the product elution is treated with a mixture Water / isopropanol / TFA in a volume ratio of 10/4 / 0.1 at a flow rate of 30-40 mL / min.
  • the flow rates were optimized for this separation accordingly. tempered and adapted.
  • a washing step with isopropanol / 0.1% TFA solution in the volume ratio 60/40 is performed.
  • the trifluoroacetic acid product fraction is added immediately after the elution in the ratio 1: 1 with a phosphate buffer (pH 10) and diluted with 15 mM sodium phosphate buffer to pH 7.2.
  • the neutralized EPO-containing solution is now filtered sterile.
  • the in-process control (IPC) for EPO enrichment is carried out on an analytical RP-HPLC separation column in the TFA / acetonitrile system.
  • the yield of EPO after this chromatography step is at least 50%, preferably at least 65% and particularly preferably at least 80%.
  • the buffer content is increased slowly, linearly from 0 to 80%.
  • the eluate fractions are analyzed and divided according to the band position in the IEF into product pool (main fraction) and marginal fractions.
  • the reference material used is a commercially available the Erypo® preparation.
  • the EPO content of the main fraction is determined by means of RP chromatography.
  • the selected eluate fractions are concentrated by ultrafiltration and rebuffered for subsequent hydroxyaptatite chromatography.
  • the yield of erythropoietin after this chromatography step is at least 20%, preferably at least 30% and particularly preferably at least 40%.
  • Isoelectric focusing is performed on an ultra-thin layer polyacrylamide gel.
  • the sample solutions must be desalted and concentrated before application with the aid of a microcentrifugation kit (cutoff 10,000 Da).
  • the focusing takes place at voltages of 300 - 2,000 V. After 5,000 Vh the development is completed.
  • the gel is then stained with silver nitrate or Coomassie and evaluated.
  • This purification step is suitable for removing the "phosphated" EPO molecules and the main fraction of the anion exchange chromatography is buffered by ultrafiltration into the starting buffer of the hydroxyapatite column and then sterile filtered.
  • the sample is injected at a rate of 30 mL / min onto a CHT Ceramic Hydroxyapatite Type 1 phase.
  • the washing step is carried out with a sodium acetate buffer (pH 6.8), the sample elution with a phosphate buffer at pH 6, 8 and a flow rate of 50 mL / min under gradient conditions.
  • the phosphate buffer content is increased slowly, linearly from 0 to 25%.
  • the specification-compliant EPO fractions are in a sodium acetate-sodium phosphate buffer and are mixed with PBS (Final completion buffer) buffered by ultrafiltration and deep-frozen at -20 0 C.
  • the EPO content of the individual fractions is determined by means of RP chromatography.
  • the yield of erythropoietin after this chromatography step is at least 30%, preferably at least 40% and particularly preferably at least 50%.
  • the analysis of the glycosylation pattern by means of HPAEC-PAD is carried out after enzymatic cleavage of the carbohydrate chains by means of PNGase F (Roche Diagnostics GmbH) from the protein. After isolation and desalination, the sugar residues are analyzed on a high-performance anion exchanger with pulsed amperometric detection.
  • the final EPO product has a biological activity of at least 150,000 IU / mg in the bio-assay and meets all the requirements of the European Pharmacopoeia

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Abstract

L'invention concerne un procédé de purification d'érythropoïétine, caractérisé en ce qu'une fraction de culture renfermant de l'érythropoïétine provenant de cellules eucaryotes produisant de l'érythropoïétine est soumise aux étapes suivantes : a) élimination des constituants cellulaires; et b) traitement du produit provenant de a) par les étapes chromatographiques ci-après, dans l'ordre donné : i) chromatographie en phase inversée; ii) chromatographie par échange d'anions; iii) chromatographie sur hydroxyapatite.
EP09757442A 2008-06-04 2009-05-28 Procédé de purification d'érytropoïétine Withdrawn EP2283036A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102008002209A DE102008002209A1 (de) 2008-06-04 2008-06-04 Verfahren zur Aufreinigung von Erythropoietin
PCT/EP2009/056544 WO2009147060A1 (fr) 2008-06-04 2009-05-28 Procédé de purification d'érytropoïétine

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EP2283036A1 true EP2283036A1 (fr) 2011-02-16

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US (1) US20110152506A1 (fr)
EP (1) EP2283036A1 (fr)
JP (1) JP2011521993A (fr)
CN (1) CN102056940A (fr)
BR (1) BRPI0914877A2 (fr)
CA (1) CA2727042A1 (fr)
DE (1) DE102008002209A1 (fr)
IL (1) IL209654A0 (fr)
WO (1) WO2009147060A1 (fr)

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IL209654A0 (en) 2011-02-28
CA2727042A1 (fr) 2009-12-10
US20110152506A1 (en) 2011-06-23
WO2009147060A1 (fr) 2009-12-10
JP2011521993A (ja) 2011-07-28
DE102008002209A1 (de) 2009-12-10
CN102056940A (zh) 2011-05-11
BRPI0914877A2 (pt) 2015-11-24

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