WO2022234412A1 - Procédé de purification de protéines de fusion fc - Google Patents

Procédé de purification de protéines de fusion fc Download PDF

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Publication number
WO2022234412A1
WO2022234412A1 PCT/IB2022/053985 IB2022053985W WO2022234412A1 WO 2022234412 A1 WO2022234412 A1 WO 2022234412A1 IB 2022053985 W IB2022053985 W IB 2022053985W WO 2022234412 A1 WO2022234412 A1 WO 2022234412A1
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Prior art keywords
chromatography
column
fusion protein
chromatography column
process according
Prior art date
Application number
PCT/IB2022/053985
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English (en)
Inventor
Ashok Kumar Mishra
Ankit Srivastava
Chaitra KACHARE
Sanjay Kumar Tiwari
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Lupin Limited
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Publication date
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Publication of WO2022234412A1 publication Critical patent/WO2022234412A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • TITLE A PROCESS FOR PURIFICATION OF FC-FUSION PROTEINS
  • the present invention relates to a process for the purification of Fc-fusion proteins. More specifically, the invention relates to a process for the purification of Fc-fusion proteins through chromatographic steps that are carried out in bind elute mode to get purified product, which is free from the process-related impurities and product- related impurities. The present invention also relates to a process for reducing the high mannose species in the purified glycoprotein.
  • Proteins are important in biopharmaceuticals as they are widely used to cure several diseases including diabetes (e.g. Insulin), cancers (e.g. Interferon, monoclonal antibodies), heart attacks, strokes, cystic fibrosis (e.g. Enzymes, Blood factors), inflammatory diseases (e.g. Tumor Necrosis Factors), anemia (e.g. Erythropoietin), hemophilia (e.g. Blood clotting factors), etc.
  • diabetes e.g. Insulin
  • cancers e.g. Interferon, monoclonal antibodies
  • heart attacks e.g. Enzymes, Blood factors
  • inflammatory diseases e.g. Tumor Necrosis Factors
  • anemia e.g. Erythropoietin
  • hemophilia e.g. Blood clotting factors
  • the protein of interest produced by the host cells during cell culture or fermentation has to be purified from certain process related impurities such as host cell-derived proteins (HCP), host-cell DNA (HCDNA), process additives; and certain product-related impurities such as degradation products, monomers, low molecular weight impurities (LMWs), and high molecular weight impurities (HMWs), oxidized species, clipped products, unfolded products, and inappropriately glycosylated protein.
  • HCP host cell-derived proteins
  • HCDNA host-cell DNA
  • product-related impurities such as degradation products, monomers, low molecular weight impurities (LMWs), and high molecular weight impurities (HMWs)
  • LMWs low molecular weight impurities
  • HMWs high molecular weight impurities
  • Fusion proteins are prepared by recombinantly expressing the genes which are created by joining two or more genes that originally code for separate proteins.
  • Fc- fusion proteins are the proteins, wherein Fc region of human Immunoglobulin G1 (IgGl) is fused with another protein of interest.
  • the active form of the Fc fusion proteins are dimers with a certain degree of glycosylation.
  • Aflibercept is a recombinant fusion protein consisting of Vascular Endothelial Growth Factor (VEGF) -binding portions from the extracellular domains of human VEGF Receptors 1 and 2 (VEGFR-1 and VEGFR-2) fused to the Fc portion of the human immunoglobulin G1 (IgGl).
  • VEGF Vascular Endothelial Growth Factor
  • Aflibercept is a dimeric glycoprotein with a protein molecular weight of 97 kilo Daltons (kDa) and contains glycosylation, constituting an additional 15% of the total molecular mass, resulting in a total molecular weight of 115 kDa.
  • Aflibercept is produced by recombinant DNA technology in a Chinese hamster ovary (CHO) K-l mammalian expression system.
  • Aflibercept is marketed as Eylea ® by Regeneron for the treatment of various ocular conditions, including wet type age related macular degeneration (AMD), and it is formulated for intravitreal administration.
  • Aflibercept is also marketed as Zaltrap ® by Sanofi- Aventis for the treatment of certain types of cancer and formulated for intravenous administration.
  • the US patent US7,070,959 describes expression and several aspects of the purification of Fc-fusion proteins including Aflibercept.
  • the US ‘759 describes process for the purification of Aflibercept, which involves Protein A chromatography using Sepharose column followed by tangential flow filtration and size exclusion chromatography.
  • Protein chromatography methods are primarily carried out in two modes i.e. flow-through mode and bind-elute mode.
  • Flow through chromatography methods rely on the property that protein of interest does not bind or binds minimally to the chromatography column and purified protein is recovered in flow through, while impurities are allowed to bind to the column.
  • bind-elute mode the protein of interest is first allowed to bind to the chromatography column under suitable conditions and then the conditions are so altered by means of suitable elution solvent or buffer system such that the bonding of the protein to the column could be reversed. Washing the column with suitable wash solvents of buffer systems allow for impurities to be separated from the protein of interest.
  • the state of art has described various approaches for purification of Fc-fusion proteins. However, these processes involve multiple chromatography steps involving various combinations of flow through and bind elute mode.
  • the Fc- protein purification processes described in the prior art involve at least one chromatography step using flow through mode.
  • the polishing chromatography steps wherein chromatography is carried out in flow through mode, the eluted protein contains impurities with similar properties. To remove such impurities in the flow through, additional multiple polishing chromatography steps are required.
  • bioreactor conditions are generally optimized to get desired level of glycosylated species in the molecule.
  • the glycosylated species do not change during typical downstream purification for any antibodies or fusion proteins.
  • Antibodies and Fc-fusion proteins bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody- dependent cell-mediated cytotoxicity (ADCC) and rapid clearance rate.
  • ADCC antibody- dependent cell-mediated cytotoxicity
  • a downstream step which reduces the high mannose species such as Man-5 and Man-6 from the product to control their level even if the cell culture produces the same in higher amount.
  • the processes described in the state of art require additional steps for obtaining desired level of glycosylation in the glycoprotein of interest. This makes these methods cumbersome, time consuming and expensive. Therefore, present inventors have developed an efficient and economically feasible process for the purification of protein which involves fewer chromatography steps, which produces protein of interest with desired glycosylation. Summary of the Invention
  • the invention relates to a process for the purification of Fc -Fusion protein comprising affinity chromatography, mixed mode chromatography and ion exchange chromatography, wherein all the chromatography steps are carried out in bind elute mode.
  • the invention in another aspect, relates to a process for the purification of Fc-fusion protein comprising: a) passing a solution comprising Fc-fusion protein and impurities through first chromatography column whereby Fc-fusion protein binds to the column, b) eluting Fc-fusion protein from first chromatography column to obtain eluate, c) passing the eluate obtained from step (b) through second chromatography column whereby Fc-fusion protein binds to the second chromatography column, d) eluting Fc-fusion protein from second chromatography column to obtain eluate e) passing eluate obtained from step (d) through third chromatography column whereby Fc-fusion protein binds to the third chromatography column, f) eluting Fc-fusion protein from third chromatography column wherein, the first chromatography is affinity chromatography wherein, the second chromatography column and the third chromatography column are selected from mixed-mode chromatography column and ion exchange chromatography column.
  • the present invention relates to a process for reducing the high mannose species in the purified glycoprotein, comprising: a) contacting glycoprotein solution containing high level of mannose species with cation exchange resin, whereby protein binds to the cation exchange resin; and b) eluting the glycoprotein from cation exchange resin to obtain glycoprotein containing lower level of high mannose species.
  • the invention relates to a process for the purification of aflibercept comprising affinity chromatography, mixed mode chromatography and ion exchange chromatography, wherein all the chromatography steps are carried out in bind elute mode.
  • Figure 1 Chromatogram of Protein A chromatography in bind elute mode performed according to present invention.
  • Figure 2 Chromatogram of Mixed mode chromatography in bind elute mode performed according to present invention.
  • Figure 3 Chromatogram of Cation exchange chromatography of in bind elute mode performed according to present invention.
  • the present invention provides a process for the purification of Fc-fusion protein comprising: a) passing a solution comprising Fc-fusion protein and impurities through first chromatography column whereby Fc-fusion protein binds to the column, b) eluting Fc-fusion protein from first chromatography column to obtain eluate, c) passing eluate obtained from step (b) through second chromatography column whereby Fc-fusion protein binds to the second chromatography column, d) eluting Fc-fusion protein from second chromatography column to obtain eluate, e) passing eluate obtained from step (d) through third chromatography column whereby Fc-fusion protein binds to the third chromatography column, f) eluting Fc -fusion protein from third chromatography column to obtain purified protein, wherein, the first chromatography column is affinity chromatography column, wherein, the second chromatography column and the third chromatography column are selected from mixed-mode chromatography column and ion
  • the Fc-Fusion protein is selected from a group comprising of but not limited to aflibercept, belatacept, rilonacept, romiplostim, abatacept, alefacept, etanercept or conbercept.
  • the Fc-fusion protein is aflibercept.
  • the affinity chromatography is carried out using Protein A or Protein G affinity chromatography.
  • the Protein A affinity chromatography is carried out using a resin selected from the group comprising of MabSelect SuReTM LX (Cytiva), MabSelectTM (GE Cytiva), and ProSep ® Ultra Plus (Millipore) resin, in a bind elute mode.
  • the Fc-fusion protein is eluted by pH gradient.
  • Affinity chromatography column is equilibrated with buffers selected from the group comprising of but not limited to Tris, Bis, and phosphate buffers.
  • affinity chromatography column is equilibrated with a Tris buffer containing sodium chloride and EDTA at a pH about 8.0 ⁇ 0.5.
  • solution containing Fc-fusion protein and impurities is loaded onto the column.
  • wash buffers in order to remove impurities from the protein. Washing of the column is carried with same or different buffers selected from Tris, Bis, acetate or phosphate buffers, preferably buffers is selected from Tris and acetate buffer.
  • the pH of the wash buffers is selected from the pH range from about 5 to about 8.
  • protein of interest is eluted from the affinity column with elution buffer by using pH gradient from pH about 5 to about 3.
  • the elution buffer is selected from acetate buffer, glycinate buffer and citrate buffer, preferably with the acetate buffer.
  • Protein A chromatography step according to present invention removes both process related impurities such as HCP, HCDNA and product related impurities such as oxidized species, HMW and LMWs.
  • the obtained protein A chromatography eluate is taken further for two polishing chromatography steps viz. mixed mode chromatography and ion exchange chromatography.
  • these two chromatography steps are carried in either order i.e. carrying out first mixed mode chromatography and then ion exchange chromatography; or first carrying out ion exchange chromatography and then mixed mode chromatography.
  • mixed mode chromatography is carried out first and then ion exchange chromatography step.
  • the pH of the eluate containing protein obtained from protein A chromatography is optionally reduced for viral inactivation and subsequently neutralized to pH about 5 before loading the eluate onto the first polishing chromatography column selected from the mixed mode chromatography and an ion exchange chromatography, preferably a mixed mode chromatography.
  • mixed mode chromatography is carried out in bind elute mode using pH gradient between pH about 9.0 to about 5.0, preferably between pH 8.0 to 6.0.
  • mixed mode chromatography is carried out using resin selected from the group comprising of PPA-HyperCelTM (propylphenyl amine) (Pall), HEA-HyperCelTM (hexylamine) (Pall), or CaptoAdhere ® (Cytiva).
  • PPA-HyperCelTM propylphenyl amine
  • HEA-HyperCelTM hexylamine
  • CaptoAdhere ® CaptoAdhere ®
  • wash buffer is selected from Tris buffer, Bis buffer and phosphate buffer at pH about 8 ⁇ 1, for efficient removal of HCP, HCDNA, HMWs and residual protein A.
  • elution buffer selected from acetate buffer, glycinate buffer, citrate buffer, phosphate buffer and preferably phosphate buffer. The elution is carried out at pH about 6.0.
  • the mixed mode chromatography column is equilibrated with 50 mM Tris having pH about 8.0 + 1 and then protein solution is loaded onto the mixed mode chromatography column. The mixed column is then washed with a Tris buffer of pH about 8.0 + 1 to remove impurities. Thereafter, protein of interest is eluted with phosphate buffer at pH about 6.0 + 1.
  • the eluate obtained from the mixed mode chromatography step is further purified by using ion exchange chromatography in a bind elute mode and pH gradient from about pH 5.0 (+ 1) to about pH 6.5 (+ 1).
  • the ion exchange chromatography according to present invention is an anion exchange chromatography or a cation exchange chromatography; preferably cation exchange chromatography.
  • cation exchange chromatography is carried out using weak cation exchange resins is selected from the group comprising of Fractogel ® COO (M) (Millipore), carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P), sulfonate(S), NuviaTM S (Bio-Rad), CaptoTM S (Cytiva) and Gigacap S (Tosoh) resins.
  • the column is equilibrated with buffers selected from Tris buffer, Bis buffer, phosphate buffer and acetate buffer. Thereafter, the eluate obtained from the mixed mode chromatography is loaded onto the cation exchange chromatography column.
  • column is optionally washed with the buffer selected from Tris buffer, Bis buffer, phosphate buffer and acetate buffer, preferably sodium phosphate buffer at pH about 6.5 (+ 0.5).
  • the cation exchange chromatography carried out according to present invention brings down the level of Man5 and Man6 species by about 50 % to about 60 % in the purified product.
  • the present invention provides to a process for the purification of aflibercept comprising: a) passing a solution comprising aflibercept and impurities through Protein A affinity chromatography column whereby aflibercept binds to protein A column, b) eluting aflibercept from protein A chromatography column to obtain first eluate, c) passing eluate obtained from step (b) through mixed mode chromatography column whereby aflibercept binds to the mixed mode chromatography column, d) eluting aflibercept from the mixed mode chromatography column to obtain second eluate e) passing the eluate obtained from step (d) through cation exchange column whereby aflibercept binds to the cation exchange column, and f) eluting aflibercept from cation exchange column to obtain purified aflibercept.
  • present invention relates to a method of treating ophthalmic diseases by administering a suitable amount of Aflibercept to a patient in need thereof wherein aflibercept is prepared according to the present invention.

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Abstract

La présente invention concerne un procédé de purification de protéines de fusion Fc, le procédé comprenant une chromatographie d'affinité suivie de deux étapes de chromatographie de polissage choisies parmi la chromatographie d'échange d'ions et la chromatographie en mode mixte, toutes les étapes de chromatographie étant effectuées dans un mode de fixation-élution. La présente invention concerne en outre la réduction d'espèces riches en mannose dans les glycoprotéines purifiées, comprenant la mise en contact d'une solution de glycoprotéine contenant des espèces riches en mannose avec une résine échangeuse de cations et l'élution de la glycoprotéine à partir de résine échangeuse de cations pour obtenir une glycoprotéine contenant un niveau inférieur d'espèces à haute teneur en mannose.
PCT/IB2022/053985 2021-05-03 2022-04-29 Procédé de purification de protéines de fusion fc WO2022234412A1 (fr)

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IN202121020258 2021-05-03
IN202121020258 2021-05-03

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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070959B1 (en) 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US20140072560A1 (en) * 2012-09-11 2014-03-13 Coherus Biosciences, Inc. Correctly folded etanercept in high purity and excellent yield
US20140323698A1 (en) 2011-11-23 2014-10-30 Didier Duthe Protein purification using bis-tris buffer
US20160083454A1 (en) 2013-05-06 2016-03-24 Sanofi Continuous multistep process for purifying antibodies
US20190119317A1 (en) * 2009-09-23 2019-04-25 E.R. Squibb & Sons, L.L.C. Cation exchange chromatography methods
US20200002373A1 (en) 2018-07-02 2020-01-02 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
US20200017544A1 (en) 2018-07-13 2020-01-16 Regeneron Pharmaceuticals, Inc. Detection and Quantification of Glycosylated Peptides
US10556942B2 (en) * 2014-06-13 2020-02-11 Lupin Limited Process for the purification of TNFR:Fc fusion protein
US20200055894A1 (en) 2018-08-17 2020-02-20 Regeneron Pharmaceuticals, Inc. Method and chromatography system for determining amount and purity of a multimeric protein
WO2020252260A1 (fr) 2019-06-13 2020-12-17 Regeneron Pharmaceuticals, Inc. Procédés d'élimination de composants indésirables pendant des processus chromatographiques en plusieurs étapes
US20210171570A1 (en) 2019-12-06 2021-06-10 Regeneron Pharmaceuticals, Inc. Anti-vegf protein compositions and methods for producing the same
WO2022129460A1 (fr) * 2020-12-18 2022-06-23 Richter Gedeon Nyrt. Procédés de purification de protéine repliée fusionnée avec un fc-peptide

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7070959B1 (en) 1999-06-08 2006-07-04 Regeneron Pharmaceuticals, Inc. Modified chimeric polypeptides with improved pharmacokinetic properties
US20190119317A1 (en) * 2009-09-23 2019-04-25 E.R. Squibb & Sons, L.L.C. Cation exchange chromatography methods
US20140323698A1 (en) 2011-11-23 2014-10-30 Didier Duthe Protein purification using bis-tris buffer
US20140072560A1 (en) * 2012-09-11 2014-03-13 Coherus Biosciences, Inc. Correctly folded etanercept in high purity and excellent yield
US20160083454A1 (en) 2013-05-06 2016-03-24 Sanofi Continuous multistep process for purifying antibodies
US10556942B2 (en) * 2014-06-13 2020-02-11 Lupin Limited Process for the purification of TNFR:Fc fusion protein
US20200002373A1 (en) 2018-07-02 2020-01-02 Regeneron Pharmaceuticals, Inc. Systems and methods for preparing a polypeptide from a mixture
US20200017544A1 (en) 2018-07-13 2020-01-16 Regeneron Pharmaceuticals, Inc. Detection and Quantification of Glycosylated Peptides
US20200055894A1 (en) 2018-08-17 2020-02-20 Regeneron Pharmaceuticals, Inc. Method and chromatography system for determining amount and purity of a multimeric protein
WO2020252260A1 (fr) 2019-06-13 2020-12-17 Regeneron Pharmaceuticals, Inc. Procédés d'élimination de composants indésirables pendant des processus chromatographiques en plusieurs étapes
US20210171570A1 (en) 2019-12-06 2021-06-10 Regeneron Pharmaceuticals, Inc. Anti-vegf protein compositions and methods for producing the same
US20210284684A1 (en) 2019-12-06 2021-09-16 Regeneron Pharmaceuticals, Inc. Anti-vegf protein compositions and methods for producing the same
WO2022129460A1 (fr) * 2020-12-18 2022-06-23 Richter Gedeon Nyrt. Procédés de purification de protéine repliée fusionnée avec un fc-peptide

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