WO2011024024A1 - Procédé de récupération d'isoformes de la darbépoïétine alpha - Google Patents

Procédé de récupération d'isoformes de la darbépoïétine alpha Download PDF

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Publication number
WO2011024024A1
WO2011024024A1 PCT/IB2009/007165 IB2009007165W WO2011024024A1 WO 2011024024 A1 WO2011024024 A1 WO 2011024024A1 IB 2009007165 W IB2009007165 W IB 2009007165W WO 2011024024 A1 WO2011024024 A1 WO 2011024024A1
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WO
WIPO (PCT)
Prior art keywords
comprised
secondary feed
mannose
galactose
darbepoetin alfa
Prior art date
Application number
PCT/IB2009/007165
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English (en)
Inventor
Villoo Morawala Patell
Sunit Maity
Sudhir Joshi
Ashutosh Vyas
Gopal Dyaga
Srinivas Ireni
Original Assignee
Avesthagen Limited
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 Avesthagen Limited filed Critical Avesthagen Limited
Publication of WO2011024024A1 publication Critical patent/WO2011024024A1/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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]

Definitions

  • the present invention relates generally to the use of novel fermentation and chromatographic procedures separately and jointly for the production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa (AVDESPTM), in biologically active form from fluids, especially mammalian host cell culture supematants.
  • AVDESPTM erythropoietin
  • AVDESPTM erythropoietin alfa
  • Anemia is often an associated condition in patients with chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • This anemia is a source of significant morbidity causing symptoms such as lack of energy, breathlessness, dizziness, angina, and poor appetite and decreased exercise tolerance.
  • the main cause of this anemia is a decreased production of erythropoietin, a naturally occurring hormone mainly produced by the kidney.
  • Much of the impaired quality of life and morbidity suffered by patients with CKD may be a consequence of this anemia and it may have a major impact on their sense of well-being as well as impairing their ability to work and affecting their social and sexual lives.
  • Recombinant human erythropoietin is currently available as a treatment for anaemia in end stage renal disease. Administration 2 to 3 times weekly is required in the majority of subjects.
  • the aim of inventing this new molecular analogue of recombinant human erythropoietin (r-HuEPO) is to obtain a therapeutic with a longer biological half-life compared to r-HuEPO, allowing a reduction of the frequency of injections necessary to maintain a desired level of systemic haemoglobin and haematocrit.
  • the chronic nature of CRF (unless a subject receives a kidney transplant) means that treatment may continue for a long part of the subject's life and multiple weekly injections of r-HuEPO can have a major impact on subjects and care givers.
  • the present invention relates generally to the use of novel chromatographic procedures separately and jointly for the production of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, in biologically active form from fluids, especially mammalian host cell culture supematants.
  • the present invention relates to the use of novel fermentation process for the overexpression of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa in CHO cells.
  • Summary of the present invention also includes use of novel chromatographic procedures for rapid and efficient recovery of novel glycoprotein possessing one or more of the biological properties of naturally occurring erythropoietin, Darbepoetin alfa from cell culture supernatant. DESCRIPTION OF FIGURE S
  • Figure 8 Western Blot Analysis of Drug substance showing comparable immuno- specificity between RMP and drug substance
  • Lane No. 1 Molecular weight
  • Lane No. 2 RMP : Reference Medicinal Product -AranespTM and Lane No. 3:
  • Figure 11 HPLC-based tryptic peptide mapping analysis.
  • Figure 12 The in vitro effect of Darbepoetin alfa has been extensively demonstrated using the reporter cell line TF1 Erythroleukamic cell assay.
  • the potency value of the sample is calculated using CFR 21/part 11 compliance with Parallel line assay (PLA) software, has shown identical profiles between RMP & Drug substance.
  • Figure 13 In Vivo Efficacy Study of Recombinant Darbepoetin Alfa for erythropoiesis stimulation in CD1 Female Mouse.
  • the present invention provides an improved process for the cell ' culture manufacturing of Recombinant Darbepoetin alfa.
  • the invention provides system that helps in achieving proper glycosylation of Recombinant Darbepoetin alfa.
  • the invention also helps in maintaining higher cell viability for a longer period of time.
  • the cell culture manufacturing process starts with seeding the bioreactor at a predefined cell density in chemically defined medium.
  • the culture is fed in two stages, primary feeding which is designed to achieve the desired cell growth and, secondary feeding which is designed to maintain the higher cell viability and hyper glycosylation of the Recombinant Darbepoetin alfa.
  • the invention also relates to bioreactor operation procedure for the manufacturing of Recombinant Darbepoetin alfa.
  • This present invention relates to the rapid and efficient recovery of Recombinant Darbepoetin alfa from cell culture supernatant from Cell culture fluid by means of Hydrophobic interaction and Ion exchange chromatography. These chromatographic steps are used for capture of recombinant Darbepoetin alfa. This separation involves in selective binding of the desired compound to specific Hydrophobic interaction resin and then elution with elution buffer. Culture supernatants are preferably clarified before chromatographic treatment.
  • Darbepoetin alfa isoforms containing eluent fractions are enriched with biologically active material, but they will be subjected to diafiltration and further processing by Anion exchange chromatographic steps.
  • the invention relates to the next chromatographic procedure where the elution from the previous step is diafiltered and further subjected to a weak cation exchange chromatographic step. These processes are used for removal of process related impurities like host cell protein and host cell DNA.
  • the active material is collected in the flow through.
  • the invention also relates to the next chromatographic procedure where the flowthrough from the previous step is diafiltered and further subjected to a strong cation exchange chromatographic step.
  • the active material is collected in the elution
  • the present invention also relates to the recombinant Darbepoetin alfa recovery procedure involving serial application different chromatographic techniques as mentioned previously. All different steps, conditions and compositions are disclosed in the invention.
  • the Bioreactor was assembled and sterilized by autoclaving at 121 0 C for 45 minutes. After sterilization, Bioreactor was charged with 7200ml of commercially available animal component free, chemically defined media. Afterwards, the bioreactor was kept under positive pressure with air at a flow rate of 0.2 Litre per minute. The bioreactor was aerated over night for 100% air saturation. The dO2 electrode was calibrated after stabilization of dissolved oxygen value. Sterile connection was created between the seed bottle and the seed port on the bioreactor head plate. The seed was then aseptically transferred to the bioreactor using peristaltic pump. The bioreactor was seeded with the density of 0.4 x 10 6 cells/mL. After seeding, the bioreactor was allowed to run at following pre-set parameters:
  • the bioreactor was sampled at every 24/48 hours for in process quality control analysis.
  • the bioreactor process was a fed - batch process with feeding of different nutrients at definite culture stages.
  • the bioreactor was daily fed with 30OmL of primary feed that comprise of glucose, galactose, mannose, lipids, amino acids, vitamins, trace elements, cholesterol and growth factors.
  • the bioreactor was daily fed with 15OmL of secondary feed that comprise of Galactose, trace Elements, Manganese Chloride and Mannose.
  • the bioreactor was operated at following pre-set and controlled parameters; pH: 7.1 ⁇ 0.1
  • the bioreactor was harvested at a cell viability of 70 - 80%.
  • the growth pattern, protein expression profile and nutrient consumptions are depicted ( Figure 1-3).
  • Clarification of the cell culture harvest was carried out by using a cellulose disposable filter with 650 - 1000 cm 2 effective filtration area and with an operating pressure of not more than 30 psi. The filtrate was checked for turbidity and target protein content.
  • the clarified harvest was diafilitered and buffer exchanged with Acetate buffer pH 4.5 - 5.5 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5 - 10 psi. 2 Molar NaCI was added to the diafiltered product.
  • Hydrophobic interaction chromatography was used in binding and elution mode with column of 100 mm diameter for capturing; with Acetate buffer pH 4.5 - 5.5 containing 2 Molar NaCI as equilibration buffer.
  • the protein of interest was eluted with Acetate buffer pH 4.5 - 5.5 ( Figure 4).
  • the HIC elute fraction was buffer exchanged with Tris buffer pH 6.8 - 7.2 using a 30 kDa - TFF membrane at a Trans Membrane Pressure of 5 - 10 psi.
  • Anion exchange chromatography in binding and elution mode was carried out with column at an operational flow rate of 10 ml/min. The column was equilibrated with Tris buffer pH 6.8 - 7.2. Protein of interest is collected in the elution.
  • This step was used for the enrichment of specific isoforms of Recombinant Darbepoetin alfa ( Figure 5). Thereafter, the elution fraction was diafiltered and buffer exchanged with Tris buffer pH 6.8-7.2. Weak cation exchange chromatography was carried out after equilibrating the column with Tris buffer pH 6.8-7.2. The Protein of interest was in the flow through. This step was used for the removal of process related impurities like host cell DNA and host cell protein (Figure 6). The eluate was buffer exchanged using a 30 kDa TFF membrane at a Trans Membrane Pressure (TMP) of 5 - 10 psi using Acetate buffer pH 3.6-4.5.
  • TMP Trans Membrane Pressure
  • the formulated material was characterized as per the specifications set by product development specification.
  • Western blot analysis for this protein showed a single diffused band corresponding to 48-50 Kda aligning with the RMP ( Figure 8).
  • the isoelectric focusing showed a pi value approximately 3.0 aligning with RMP.
  • This drug substance when analysed in Reverse Phase HPLC showed the retention time of 15.222 minutes with the test sample in comparison to RMP value of 15.350 minutes( Figure 9).
  • Size Exclusion HPLC also revealed retention time of 8.800 minutes in comparison to the retention time 8.767 of RMP.
  • Intact molecular mass estimation performed by high- sensitivity MALDI-TOF MS analysis has revealed the molecular mass of purified Darbepoetin alfa and RMP to be 37.5 kDa ( Figure 10).
  • Acute Intravenous Toxicity and Subcutaneous Toxicity Study of Darbepoetin alfa in Wistar Rat were carried out with a human equivalent dose of 1OX.
  • the same study of intravenous and subcutaneous was also carried out in the similar way in Swiss Albino mice. From the studies, inference drawn were that no clinical signs of toxicity was observed at single dose administration of Darbepoetin Alfa by intravenous and subcutaneous routes in rat and mouse.
  • In Vivo Efficacy Study of Darbepoetin Alfa in CD1 mice for erythropoiesis stimulation was carried out.
  • test substance did not produce any significant adverse effect even at high dose i.e. 15.5 mcg/kg for rabbit and 31 mcg/kg for rat.
  • a Repeated Dose Developmental toxicity study of biosimilar Darbepoetin Alfa in pregnant Wistar Rats was also carried out. Based on the findings, inference was that the test substance did not produce any significant adverse effect on repeated dose during gestation period to the dams as well as the pups. Further study on skin sensitization potential also inferred that Darbepoetin alfa is a non-sensitizer.

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

Abstract

La présente invention a pour objet l'utilisation de nouvelles techniques de fermentation et chromatographiques séparément et conjointement pour la production de la protéine darbépoïétine alpha recombinante, sous une forme biologiquement active à partir de liquides, spécialement des surnageants de cultures de cellules hôtes de mammifères.
PCT/IB2009/007165 2009-08-28 2009-10-20 Procédé de récupération d'isoformes de la darbépoïétine alpha WO2011024024A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN2074/CHE/2009 2009-08-28
IN2074CH2009 2009-08-28

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WO2011024024A1 true WO2011024024A1 (fr) 2011-03-03

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013058485A1 (fr) 2011-10-18 2013-04-25 Chong Kun Dang Pharmaceutical Corp. Procédés de purification d'analogues d'érythropoïétine ayant un point isoélectrique plus faible
KR101414897B1 (ko) * 2013-11-29 2014-07-04 씨제이헬스케어 주식회사 다베포에틴 알파의 정제 방법
WO2017154869A1 (fr) * 2016-03-09 2017-09-14 Ｊｃｒファーマ株式会社 Procédé de production d'érythropoïétine humaine mutante
WO2023040792A1 (fr) * 2021-09-14 2023-03-23 杰科（天津）生物医药有限公司 Procédé de préparation d'érythropoïétine

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006126066A2 (fr) * 2005-05-24 2006-11-30 Avestha Gengraine Technologies Pvt Ltd. Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese
US7217689B1 (en) * 1989-10-13 2007-05-15 Amgen Inc. Glycosylation analogs of erythropoietin
US20070161084A1 (en) * 2005-12-08 2007-07-12 Amgen Inc. Production of glycoproteins using manganese
WO2007136752A2 (fr) * 2006-05-19 2007-11-29 Glycofi, Inc. Compositions d'érythropoïétine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7217689B1 (en) * 1989-10-13 2007-05-15 Amgen Inc. Glycosylation analogs of erythropoietin
WO2006126066A2 (fr) * 2005-05-24 2006-11-30 Avestha Gengraine Technologies Pvt Ltd. Methode recombinante pour la production d'une proteine de stimulation de l'erythropoiese
US20070161084A1 (en) * 2005-12-08 2007-07-12 Amgen Inc. Production of glycoproteins using manganese
WO2007136752A2 (fr) * 2006-05-19 2007-11-29 Glycofi, Inc. Compositions d'érythropoïétine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WERNER R.G. ET AL: "Glycosylation of therapeutic proteins in different production systems", ACTA PAEDIATRICA, vol. 96, 2007, pages 17 - 22, XP008080645 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013058485A1 (fr) 2011-10-18 2013-04-25 Chong Kun Dang Pharmaceutical Corp. Procédés de purification d'analogues d'érythropoïétine ayant un point isoélectrique plus faible
EP2768846A4 (fr) * 2011-10-18 2015-05-06 Chong Kun Dang Pharm Corp Procédés de purification d'analogues d'érythropoïétine ayant un point isoélectrique plus faible
KR101414897B1 (ko) * 2013-11-29 2014-07-04 씨제이헬스케어 주식회사 다베포에틴 알파의 정제 방법
WO2015080509A1 (fr) * 2013-11-29 2015-06-04 씨제이헬스케어 주식회사 Procédé de purification de darbépoétine alfa
CN105764915A (zh) * 2013-11-29 2016-07-13 Cj医药健康株式会社 纯化达依泊汀α的方法
JP2016538261A (ja) * 2013-11-29 2016-12-08 シージェイ ヘルスケア コーポレイションＣｊ Ｈｅａｌｔｈｃａｒｅ Ｃｏｒｐｏｒａｔｉｏｎ ダルベポエチンアルファの精製方法
US10723775B2 (en) 2013-11-29 2020-07-28 Cj Healthcare Corporation Method for purifying darbepoetin alfa
WO2017154869A1 (fr) * 2016-03-09 2017-09-14 Ｊｃｒファーマ株式会社 Procédé de production d'érythropoïétine humaine mutante
US20190078129A1 (en) * 2016-03-09 2019-03-14 Jcr Pharmaceuticals Co., Ltd. Method for production of mutant-type human erythropoietin
US10604779B2 (en) 2016-03-09 2020-03-31 Jcr Pharmaceuticals Co., Ltd. Method for production of mutant-type human erythropoietin
WO2023040792A1 (fr) * 2021-09-14 2023-03-23 杰科（天津）生物医药有限公司 Procédé de préparation d'érythropoïétine

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