WO2020234742A1 - Purification du facteur de stimulation de colonies de granulocytes - Google Patents

Purification du facteur de stimulation de colonies de granulocytes Download PDF

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Publication number
WO2020234742A1
WO2020234742A1 PCT/IB2020/054680 IB2020054680W WO2020234742A1 WO 2020234742 A1 WO2020234742 A1 WO 2020234742A1 IB 2020054680 W IB2020054680 W IB 2020054680W WO 2020234742 A1 WO2020234742 A1 WO 2020234742A1
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WO
WIPO (PCT)
Prior art keywords
gcsf
anion exchange
host cell
flow
exchange chromatography
Prior art date
Application number
PCT/IB2020/054680
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English (en)
Inventor
Sandeep Suresh SOMANI
Original Assignee
Lupin Limited
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Publication date
Application filed by Lupin Limited filed Critical Lupin Limited
Publication of WO2020234742A1 publication Critical patent/WO2020234742A1/fr

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF

Definitions

  • the present invention relates to a method for purification of recombinant human granulocyte colony stimulating factor (rh-GCSF) from one or more process and product related impurities by using an anion exchange chromatography, operated in flow-through mode.
  • rh-GCSF human granulocyte colony stimulating factor
  • G-CSF is a glycoprotein that stimulates differentiation of progenitor“stem cells” into granulocytes.
  • G-CSF has been used in treatment of neonatal infections, neutropenia, and to tackle severe infections and sepsis in acute myeloid leukaemia's.
  • Recombinant human Granulocyte cell stimulating factor (rh-GCSF) has been therapeutically used and is indicated for treatment of chemotherapy induced neutropenia in cancer patients.
  • GCSF is one of several protein therapeutics which are produce by recombinant DNA technology.
  • Recombinant GCSF produced in both bacteria (Filgrastim) and mammalian cells (Lenograstim) are clinically available.
  • Recombinant Filgrastim, produced in bacteria, such as E.coli. are usually expressed in the form of inclusion bodies. Therefore, preparation of therapeutically effective Filgrastim involves processes, such as inclusion bodies isolation, solubilization in the presence of protein denaturing agents, followed by refolding the protein to its native state. The refolded rh-GCSF is then purified by various chromatographic approaches.
  • Filgrastim produced in E.coli has a molecular weight of about 18Kda, with an isoelectric point of about 5.5.
  • U.S. patents 4810643, 4999291, 5055555, 5849883, 5582823, 5580755 and 5830705 describe various aspects of recombinant expression and purification of the h-GCSF protein from various expression systems ranging from bacterial cells to yeast and mammalian cells. Expression of rhG-CSF as inclusion bodies in a bacterial system is described in U.S. Patent 4810643. Recombinant protein production always leads to two broad classes of impurities - Process related and Product related.
  • Process related impurities primarily constitute of impurities such as host cell DNA (HCDNA), host cell proteins (HCP), bacterial endotoxins (BET), while impurities, such as high molecular weight aggregates (HMW) or low molecular weight variants (LMW), constitute product related impurities.
  • impurities such as host cell DNA (HCDNA), host cell proteins (HCP), bacterial endotoxins (BET), while impurities, such as high molecular weight aggregates (HMW) or low molecular weight variants (LMW) constitute product related impurities.
  • HMW high molecular weight aggregates
  • LMW low molecular weight variants
  • Chromatography is primarily carried out in two modes i.e. bind-elute mode and flow-through mode.
  • a bind-elute mode i.e. 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 post. Washing the column with suitable wash solvents of buffer systems allow for impurities to be separated from the protein of interest. Contrary to a bind elute mode chromatography, flow-through chromatography relies on the property that the protein of interest does not bind or binds minimally to the column and purified protein is recovered in flow-through, while the impurities are allowed to bind to the column.
  • US5849883 discloses purification of refolded bovine GCSF by using an ion exchange chromatography. Specifically, US ‘883 utilizes a bind elute mode chromatography using a CM Sepharose IEX column. US6489447 broadly describes protein purification using an ion exchange chromatography in bind-elute mode, and particularly attempts to address separation of deamidated variants. It also describes the importance of changing wash buffer conductivity and/or pH in protein purification. US9453045 discloses use of multimodal chromatography for purification of recombinant GCSF obtained from culture supernatant.
  • the main aim of instant invention is to develop a fast and cost-effective flow-through based process for purification of rh-GCSF, which is effective at removal of various process related impurities, such as HCP and HCD as well as product related impurities such as HMW species of refolded a rh-GCSF.
  • process related impurities such as HCP and HCD
  • product related impurities such as HMW species of refolded a rh-GCSF.
  • the present invention provides a method of purifying GCSF using anion exchange chromatography for impurities removal.
  • the invention provides a method for purifying refolded rh-GCSF using an anion exchange chromatography in flow-through mode.
  • the invention provides a method for purifying GCSF comprising the steps:
  • the refolded GCSF is loaded onto anion exchange resin with a buffer at conductivity not less than 4.50 mS/cm and pH about 7.1 -7.8.
  • the refolded GCSF may be subjected to ultrafiltration/Diafiltration before loading onto anion exchange column.
  • the anion exchange chromatographic step may be preceded by a tangential flow filtration step.
  • the invention provides a method of purification of GCSF by anion exchange chromatography, wherein the anion exchange flow-through is collected as a whole or as a fraction from the anion exchange resin.
  • the impurity removal using anion exchange chromatography is more that 70%.
  • the invention provides a method for purifying rh-GCSF comprising following steps: a) recovering the refolded GCSF from inclusion bodies produced in the recombinant expression
  • anion exchange chromatography step removes about greater than 70% impurities.
  • the anion exchange chromatography step may be followed by another chromatographic step.
  • the invention provides a method for purifying GCSF comprising the following steps a) isolating the inclusion bodies of GCSF bacterial host cell;
  • step (f) collecting the purified GCSF in flow-through from the anion exchange column in step (f) wherein the anion exchange chromatography step is capable of removing at least 90% of host cell proteins (HCP), host cell DNA (HCDNA), bacterial endotoxin (BET), and high molecular aggregates.
  • HCP host cell proteins
  • HCDNA host cell DNA
  • BET bacterial endotoxin
  • the embodiments mentioned herein may further include one or more sterile filtration or nano filtration steps.
  • the invention provides a method for removal of process and product related impurities from a GCSF using anion exchange chromatography, operated in flow through mode.
  • the present invention provides a method of purification of GCSF from one or more impurities using anion exchange chromatography.
  • the invention provides a method of purification of an GCSF from a sample comprising one or more impurities, comprising a) loading the sample onto an anion exchange chromatographic resin b) collecting the flow through.
  • the invention describes a method for purification of refolded GCSF using anion exchange chromatography, wherein HCP, HCDNA, BET and HMW are reduced by at least 90%.
  • GCSF or G-CSF or rh-GCSF has been used interchangeably herein, refers to recombinant human granulocyte colony stimulating factors or its analogues with isoelectric point is 5.5+ 1.
  • chaotropic agent(s) refers to a compound that, in a suitable concentration in aqueous solution, is capable of changing the spatial configuration or conformation of polypeptides through alterations at the surface thereof so as to render the polypeptide soluble in the aqueous medium.
  • chaotropic agents include guanidine hydrochloride, urea, and hydroxides such as sodium or potassium hydroxide. Chaotropic agents may also include a combination of these reagents, such as a mixture of a hydroxide with urea or guanidine hydrochloride.
  • reducing agent(s) refer to a compound that, at a suitable concentration in aqueous solution, maintain the free sulfhydryl groups, such that intra- or intermolecular disulfide bonds are chemically disrupted.
  • suitable reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), cysteine, cysteamine, thioglycolate, glutathione, and sodium borohydride.
  • buffered solution refers to a solution which resists changes in pH by the action of its acid-base conjugate components.
  • solubilization buffer refers to a solution used for solubilization of inclusion bodies.
  • refolding solution refers to a solution used for recovering a desire refolded protein confirmation from solubilized inclusion bodies.
  • Flow-through mode refers to a chromatography process, wherein the protein on interest is allowed to flow though the column during the loading of the column as obtained as part of the unbound or "flow-through” fraction during loading or post load wash of the chromatography resin.
  • Anion exchange resin refers to a solid chromatographic support, which has a positively charged ligand such as a quaternary amino group attached thereto, capable of ionic interaction with a negatively charged protein or a functional group under suitable conditions.
  • the anion exchange resin can be any weak or strong anion exchange resin or a membrane which could function as a weak or a strong anion exchanger.
  • anion exchange resins include without any limitation DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE Healthcare, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Q-Ceramic Hyper D, DEAE-Ceramic Hyper D, from Pall Corporation.
  • purification or “purifying” or fractionation has been used interchangeably herein, refer to increasing the degree of purity of a protein of interest in particular rh-GCSF, from a sample or a preparation comprising the refolded rh- GCSF and one or more process or product related impurities.
  • impurity refers to any proteinaceous or non-proteinaceous molecular entity distinct from the than the protein of interest.
  • the impurity includes, without limitation: host cell protein, host cell DNA, fragment, aggregate, another polypeptide, endotoxin, bacterial cell culture media component etc.
  • the protein of interest may be described as the fraction of protein, substantially free of impurities, as described herein, and can be obtained as part of the flow through from the anion exchange chromatography.
  • Solubilization and refolding of GCSF has already been disclosed in WO2010/146599.
  • inclusion bodies (IBs)of rh-GCSF were solubilized in lOOmM Tris, 6M GuHCl at pH 8.0 over a period of around 45 min.
  • the OD of the solubilized IB was adjusted with solubilization buffer to 8.0.
  • the solution containing solubilized inclusion bodies was filtered through 0.45pm filter. DTT was added up to 5mM to reduce the protein. Reduction was carried out for 30 min at room temperature (25 °C).
  • the solubilized GCSF was added to a refolding buffer, containing 75mM Tris pH 8.8, 0.1M L- Arginine, 10% sucrose, 2mM EDTA, lOmM Sodium ascorbate, 2M Urea, and stirred over a period of 30-45 minutes.
  • Refolding buffer temperature of the buffer was maintained at around 8.0°C, and refolding was continued for a period of 15- 20 hrs.
  • sodium ascorbate is used in refolding buffer dehydro ascorbate and reduced glutathione are also added in refolding buffer to provide redox condition while refolding.
  • oxido- shuffling agents such as Cysteine/Cystine or Oxidised and reduced glutathione can also be used.
  • the solution was buffer exchanged to 20mM Tris pH 8.0, 5% Sucrose or 5% D + Sorbitol and the denaturant was removed.
  • Example 3 Size exclusion HPLC (SE-HPLC) and Reverse Phase HPLC (RP- HPLC)

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne la purification de protéines pour l'élimination d'impuretés associées à un produit et à un procédé. En particulier, l'invention concerne la purification du facteur de stimulation des colonies de granulocytes humain de recombinaison (rh-GCSF) à l'aide d'une chromatographie d'échange d'anions dans un mode d'écoulement continu pouvant éliminer au moins 90 % de protéines de cellules hôtes (HCP), d'ADN de cellules hôtes (HCDNA), d'endotoxine bactérienne (BET) et d'agrégats de haut poids moléculaire (HMW).
PCT/IB2020/054680 2019-05-20 2020-05-18 Purification du facteur de stimulation de colonies de granulocytes WO2020234742A1 (fr)

Applications Claiming Priority (2)

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IN201921019886 2019-05-20
IN201921019886 2019-05-20

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WO2020234742A1 true WO2020234742A1 (fr) 2020-11-26

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022167886A1 (fr) * 2021-02-04 2022-08-11 Intas Pharmaceuticals Ltd. Procédé amélioré de purification de peg-gcsf à double ufdf

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4810643A (en) 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
WO1989010932A1 (fr) * 1988-05-13 1989-11-16 Amgen Inc. Compositions et procede de traitement ou de prevention d'infections chez des animaux
US4999291A (en) 1985-08-23 1991-03-12 Amgen Inc. Production of human pluripotent granulocyte colony-stimulating factor
US5055555A (en) 1989-01-05 1991-10-08 Helmut Sassenfeld Purification of G-CSF
US6489447B1 (en) 1998-05-06 2002-12-03 Genentech, Inc. Protein purification
WO2006061851A2 (fr) * 2004-12-09 2006-06-15 Zenotech Laboratories Limited Procede pour la production d'un facteur stimulant les colonies de granulocytes-macrophages
WO2010146599A1 (fr) 2009-06-16 2010-12-23 Lupin Limited Procédé de purification du facteur de croissance hématopoïétique humain de recombinaison
WO2012057529A2 (fr) * 2010-10-29 2012-05-03 Hanmi Holdings Co., Ltd. Procédé pour purifier un facteur stimulant les colonies de granulocytes humain de e. coli recombinant
WO2013068602A2 (fr) * 2012-03-19 2013-05-16 Richter Gedeon Nyrt. Procédé de production de polypeptides
US9453045B2 (en) 2010-03-30 2016-09-27 Octapharma Ag Process for the purification of a growth factor protein

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4999291A (en) 1985-08-23 1991-03-12 Amgen Inc. Production of human pluripotent granulocyte colony-stimulating factor
US4810643A (en) 1985-08-23 1989-03-07 Kirin- Amgen Inc. Production of pluripotent granulocyte colony-stimulating factor
US5580755A (en) 1985-08-23 1996-12-03 Amgen Inc. Human pluripotent granulocyte colony-stimulating factor
US5582823A (en) 1985-08-23 1996-12-10 Amgen Inc. Methods of treating bacterial inflammation and granulocytopoiesis by administering human pluripotent granulocyte colony-stimulating factor
US5830705A (en) 1985-08-23 1998-11-03 Amgen Inc. Method for recombinant production of human pluripotent granulocyte colony-stimulating factor
US5849883A (en) 1988-05-13 1998-12-15 Amgen Inc. Method for purifying granulocyte colony stimulating factor
WO1989010932A1 (fr) * 1988-05-13 1989-11-16 Amgen Inc. Compositions et procede de traitement ou de prevention d'infections chez des animaux
US5055555A (en) 1989-01-05 1991-10-08 Helmut Sassenfeld Purification of G-CSF
US6489447B1 (en) 1998-05-06 2002-12-03 Genentech, Inc. Protein purification
WO2006061851A2 (fr) * 2004-12-09 2006-06-15 Zenotech Laboratories Limited Procede pour la production d'un facteur stimulant les colonies de granulocytes-macrophages
WO2010146599A1 (fr) 2009-06-16 2010-12-23 Lupin Limited Procédé de purification du facteur de croissance hématopoïétique humain de recombinaison
US9453045B2 (en) 2010-03-30 2016-09-27 Octapharma Ag Process for the purification of a growth factor protein
WO2012057529A2 (fr) * 2010-10-29 2012-05-03 Hanmi Holdings Co., Ltd. Procédé pour purifier un facteur stimulant les colonies de granulocytes humain de e. coli recombinant
WO2013068602A2 (fr) * 2012-03-19 2013-05-16 Richter Gedeon Nyrt. Procédé de production de polypeptides

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WILLIAM E. COLLINS: "Protein Separation with Flow-Through Chromatography", SEPARATION AND PURIFICATION METHODS., vol. 26, no. 2, 1 January 1997 (1997-01-01), US, pages 215 - 253, XP055709095, ISSN: 0360-2540, DOI: 10.1080/03602549708014159 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022167886A1 (fr) * 2021-02-04 2022-08-11 Intas Pharmaceuticals Ltd. Procédé amélioré de purification de peg-gcsf à double ufdf

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