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WO2011156369A2 - Purification de cytokines modifiées - Google Patents

Purification de cytokines modifiées Download PDF

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Publication number
WO2011156369A2
WO2011156369A2 PCT/US2011/039439 US2011039439W WO2011156369A2 WO 2011156369 A2 WO2011156369 A2 WO 2011156369A2 US 2011039439 W US2011039439 W US 2011039439W WO 2011156369 A2 WO2011156369 A2 WO 2011156369A2
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WO
WIPO (PCT)
Prior art keywords
chromatography
process according
isoforms
hydrophobic anion
mixture
Prior art date
Application number
PCT/US2011/039439
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English (en)
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WO2011156369A3 (fr
Inventor
Darshan Koticha
Kishore Jahagirdhar
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Dr. Reddy's Laboratories Ltd.
Dr. Reddy's Laboratories, Inc.
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Publication date
Application filed by Dr. Reddy's Laboratories Ltd., Dr. Reddy's Laboratories, Inc. filed Critical Dr. Reddy's Laboratories Ltd.
Publication of WO2011156369A2 publication Critical patent/WO2011156369A2/fr
Publication of WO2011156369A3 publication Critical patent/WO2011156369A3/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/32—Bonded phase chromatography
    • B01D15/325—Reversed phase
    • B01D15/327—Reversed phase with hydrophobic interaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange

Definitions

  • aspects of the present application relate to methods for purifying cytokines. Specific aspects relate to methods for purifying darbepoetin alpha.
  • the application relates to methods for separating darbepoetin alpha isoforms, comprising hydrophobic anion induction chromatography.
  • Erythropoiesis stimulating proteins such as erythropoietin and analogs of erythropoietin, are glycoprotein hormones that are the principle homeostatic regulators of red blood cell production. Though natural erythropoietin is produced by the kidney, its large scale production for therapeutic purposes is achieved by recombinant DNA methods. Purified recombinant human
  • erythropoietin is administered in human patients for the treatment of medical indications associated with inadequate red blood cell supply, e.g., anemia and chronic renal failure.
  • rHuEPO and its analog darbepoetin alpha are used for the treatment of anemia and disrupted red blood cell production associated with various conditions, such as perisurgery, chronic renal failure, as well in the treatment of side effects associated with HIV, HCV and cancer chemotherapy.
  • Negatively charged sialic acid residues typically cap the ends of a glycan chain of most glycoproteins.
  • Human erythropoietin and darbepoetin alpha expressed in Chinese hamster ovary (CHO) cells exhibit variable degrees of glycosylation and sialylation. Variability in the glycan chains, in particular in the sialic acid content, results in erythropoietin and darbepoetin alpha isoforms that differ in their overall charge and isoelectric point (R. S. Rush et al., Analytical Chemistry, Vol. 67, pages 1442-1452, 1995).
  • erythropoietin and darbepoetin alpha isoforms with higher sialic content have lower isoelectric point (pi), than those with lower sialic acid content.
  • Variability in the extent of sialylation in turn effects the in vivo activity of erythropoietin and darbepoetin compositions.
  • Compositions enriched in low pi isoforms exhibit higher bioactivity then those with higher content of high pi isoforms (Takeuchi et al., 1989 PNAS
  • darbepoetin alpha exhibit higher specific activity than isoforms with higher pi.
  • darbepoetin alpha isoforms with lower pi are of greater therapeutic value.
  • Aranesp® Amgen
  • the approved and marketed form of darbepoetin alpha comprises essentially of low pi isoforms of the protein, having a pi range of 3-3.9 (Francoise Lasne et al., Analytical Biochemistry 311 (2002) 119-126).
  • darbepoetin alpha is expressed in cell culture as a heterogeneous mixture of isoforms in the pi range of about 3 to 8. Hence, there is a need for efficient and effective methods for the separation of low pi isoforms from higher pi isoforms of darbepoetin alpha.
  • erythropoietin and analogues of erythropoietin.
  • WO 03045996 discloses chromatographic purification of recombinant human erythropoietin by reverse phase chromatography, anion exchange and size exclusion chromatography.
  • aspects of the present application provide processes for separating and purifiying low pi isoforms of darbepoetin alpha from mixtures of isoforms, comprising hydrophobic anion induction chromatography.
  • Fig. 1 is a chromatography profile of hydrophobic anion induction chromatography as performed in Example 4.
  • Fig. 2 is an isoelectric focusing gel of the hydrophobic anion induction chromatography performed as described in Example 4.
  • Fig. 3 shows a comparison of darbepoetin alpha samples from different batches according to Example 4 with a commercially available darbepoetin alpha product, Aranesp® (pi 3-3.9, Amgen). DETAILED DESCRIPTION
  • Darbepoetin alpha is an erythropoietin analog with five N-linked
  • darbepoetin alpha exhibits a three-fold longer serum half-life and increase in vivo activity as compared to recombinant human EPO.
  • darbepoetin alpha is expressed as a heterogeneous mixture of low and high pi isoforms that differ in their extent of glycosylation and sialylation.
  • low pi isoforms exhibit much higher specific activity as compared to isoforms of higher pi having lower sialic acid content (e.g. Imai et al., European Journal of Biochemistry, 194 (1990), 457-462; European Patent Application Publication 0 428 267).
  • isoforms with higher sialic acid content and lower pi are of greater therapeutic value.
  • aspects of the present application provide efficient methods for the isolation and separation of darbepoetin alpha isoforms from a mixture comprising low and high pi isoforms, using hydrophobic anion induction chromatography.
  • Hydrophobic anion induction chromatography is a type of dual mode or mixed mode chromatography.
  • Mixed mode chromatography exploits the unique properties of certain chromatographic ligands that show either hydrophobic or charged interactions, or both (Boschetti et al, Gen. Engineering News, 20, 2000; Ghose et al, Biotechnoogy Progress, 21, 498 - 508, 2005, for detailed discussions of mixed mode chromatography).
  • Hydrophobic anion induction chromatography sorbents possess chromatographic ligands that are hydrophobic at one pH and positively charged at a lower pH. Hence, such chromatographic ligands contribute hydrophobic characteristics to the chromatographic resin at higher pH values and anion exchange characteristics at lower pH values.
  • MEP HyperCelTM 4-mercaptoethyl pyridine (MEP).
  • MEP has a pK a of 4.8 and the nitrogen atom in its pyridine ring acquires a positive charge at low pH.
  • MEP HyperCel acts as a hydrophobic interaction chromatography medium, and at low pH, as an anion exchange chromatography medium.
  • Hydrophobic anion induction chromatography has been used for the purification of monoclonal antibodies, wherein the antibodies are bound to the column at close to neutral pH. Under acidic conditions, both, the chromatographic ligand and the bound antibody take on a net positive charge. Binding is thus disrupted and elution occurs (Schwartz et al, Journal of Chromatography A, 908, 251-263 21, 2001).
  • hydrophobic anion induction chromatography is used for the separation of low pi isoforms from a mixture comprising low and high pi isoforms of darbepoetin alpha.
  • the application provides methods for the separation of low pi isoforms of darbepoetin alpha from a solution comprising low and high pi isoforms by hydrophobic anion induction chromatography, comprising:
  • the loading solution has pH values about 3 to about 4, In embodiments, the loading solution has pH 3.3.
  • the conductivity of the loading solution may be less than or equal to 0.3 mS/cm.
  • the elution buffer has pH values greater than about 4.5. In embodiments, the elution buffer has pH values between about 5 and about 6.
  • the elution buffer has pH about 6.
  • the conductivity of the elution buffer is about 4 mS/cm to about 30 mS/cm.
  • the conductivity of the elution buffer is about 10 mS/cm to about 20 mS/cm.
  • the conductivity of the elution buffer is about 15 mS/cm.
  • the present application provides methods for the separation of low pi isoforms of darbepoetin alpha from a solution comprising low and high pi isoforms by hydrophobic anion induction chromatography, comprising loading the solution onto the hydrophobic anion induction chromatography resin at pH about 3.3 and conductivity about 0.3 mS/cm, and eluting the low pi isoforms at pH about 6 and conductivity about 15 mS/cm.
  • the hydrophobic anion induction chromatography may be preceded by ion exchange chromatography.
  • the hydrophobic anion induction chromatography may be preceded by two ion exchange chromatography.
  • the hydrophobic anion induction chromatography may be preceded and followed by ion exchange chromatography.
  • the hydrophobic anion induction chromatography may be preceded by an anion exchange chromatography.
  • the hydrophobic anion induction chromatography may be preceded by an anion exchange chromatography and a cation exchange chromatography.
  • the hydrophobic anion induction chromatography may be preceded by an affinity chromatography.
  • the embodiments mentioned here may include one or more of viral inactivation, sterile filtration, and viral filtration steps.
  • a mixed-mode chromatography sorbent such as MEP
  • MEP HyperCelTM (Pall Corporation, USA) is used.
  • MEP HyperCel sorbent is a 80-100 ⁇ particle size high porosity cross-linked cellulose matrix having 80-125 ⁇ /nnL of a 4-mercaptoethyl pyridine (4-MEP) ligand.
  • affinity chromatography using materials such as Blue SepharoseTM 6 Fast Flow (GE Healthcare Life Sciences) or a Prosep®-PB (Millipore) is used.
  • Blue Sepharose 6 Fast Flow resin is made using a Cibacron Blue 3G coupled to Sepharose 6 Fast Flow, which is a highly cross- linked 6% agarose matrix.
  • ProSep-PB media is composed of a synthetic m- aminophenyl ligand (phenyl boronate ligand) immobilized on controlled pore size glass beads.
  • Anion exchange chromatography in embodiments may be carried out using any weak or strong anion exchange chromatographic resin, or a membrane which can function as a weak or a strong anion exchanger.
  • anion exchange resins include, but are not limited to, DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied Biosystems, MonoQ, MiniQ, Source 15Q and 3OQ, Q, DEAE and ANX Sepharose Fast Flow, Q Sepharose high Performance, QAE SEPHADEX and FAST Q SEPHAROSE from GE
  • a strong anion exchange resin such as Q-Sepharose Fast FlowTM (GE Healthcare Life Sciences) is used. This resin is made using a highly cross- linked 6% agarose matrix attached to a -O-CH2CHOHCH2OCH2CHOHCH2- N + (CH 3 )3 functional group.
  • Cation exchange chromatographic steps in embodiments may be carried out using any weak or strong cation exchange chromatographic resin, or a membrane which could function as a weak or a strong cation exchanger.
  • cation exchange resins include, but are not limited to, those having a sulfonate based group, e.g., MonoS, MiniS, Source 15S and 30S, SP Sepharose Fast Flow, SP Sepharose High Performance from GE Healthcare, Toyopearl SP-650S and SP-650M from Tosoh, S-Ceramic Hyper D, from Pall Corporation or a carboxymethyl based group e.g., CM Sepharose Fast Flow from GE Healthcare, Macro-Prep CM from BioRad, CM-Ceramic Hyper D, from Pall Corporation, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh.
  • a sulfonate based group e.g., MonoS, MiniS, Source 15S and 30S
  • SP Sepharose Fast Flow SP Sepharose High Performance from GE Healthcare
  • Toyopearl SP-650S and SP-650M from Toso
  • a weak cation exchange column such as CM - SepharoseTM (GE Healthcare Life Sciences) is used.
  • This resin is made using a highly cross-linked 6% agarose matrix attached to a carboxymethyl group.
  • the buffering agents used for making buffer solutions may comprise acetate or phosphate buffers, including any of their salts or derivatives.
  • isoform refers to proteins with identical amino acid sequences, but differing with respect to charge and therefore isoelectric points, as a result of differences in glycosylation, acylation, deamidation, or sulfation.
  • the "isoelectric point” or “pi” is the pH at which a particular molecule or surface carries no net electrical charge.
  • the "pi” of a polypeptide refers to the pH at which the polypeptide's positive charge balances its negative charge.
  • the pi can be estimated by various methods known in the art, e.g., from the net charge of the amino acid and/or sialic acid residues on the polypeptide, or by using isoelectric focusing, chromatofocussing, etc.
  • the low pi isoforms refer to isoforms with pi of 4 or less.
  • the pooled population of cells is diluted to very low cell density (1 -3 viable cells/200 ⁇ _ media) and plated in 96 well microtiter plates to establish clonal cell lines that originate from single cells. Clones are screened for darbepoetin alpha production and clones with high productivity are selected for expression.
  • the cells expressing darbepoetin alpha are expanded from the master cell bank a stages of spinners and one stage of seed reactor before being inoculated into the production reactor.
  • PF-CHO medium is used for culturing the cells in spinners in order to obtain good cell growth and high viability.
  • the PF-CHO medium contains, per liter of medium: PF-CHO main powder 6.0 g, PF-CHO base powder 10.4 g, L- Glutamine 0.58 g, Pluronic F-68 1 .0 g, sodium bicarbonate 2.0 g.
  • the pH of the medium is adjusted to 7 before inoculation.
  • Cells from the master cell bank are inoculated in a spinner bottle containing PF-CHO medium at an initial cell count of 0.2 million cells/mL.
  • the spinner bottles are incubated in a 5% CO2 incubator maintained at 37°C.
  • SFM-6(1 ) medium contains, "DMEM/F-12" basal media, amino acids, insulin, vitamins, trace elements, plant peptone, bicarbonate, and fructose sugar.
  • the pH is the pH of the seed reactor.
  • the culture is aseptically harvested and cells are transferred to a 10 L production reactor containing 9 L of SFM-6(2) medium at an initial cell density of 0.2 million cells/mL. The culture is harvested after 12 days to collect the supernatant liquid containing the desired product.
  • the cell culture broth from the Expression and Harvest of Protein procedure described above is concentrated and the conductivity is reduced by diafiltration (using tangential flow filtration (TFF) with a molecular weight cut off of 30 kDa) using 25 mM Tris, 60 mM NaCI buffer having pH 7.1 .
  • the concentrated cell culture broth is then loaded onto a Q-Sepharose column (300 ml_, XK 50/20) that was pre-equilibrated with 5 column volumes (CV) of 25 mM Tris, 60 mM NaCI, pH 7.1 buffer.
  • the column is then washed with 5 CV of the equilibration buffer (25 mM Tris, 60 mM NaCI, pH 7.1 ).
  • Eluate from Example 1 containing the desired protein, is concentrated and exchanged with buffer containing 83.4 mM sodium acetate (or 80-85 mM sodium acetate) pH 3.3 (or pH 3-3.5) using TFF.
  • Sample from TFF is loaded onto a MEP chromatographic column (4 ml_, Tricorn 5/20) pre-equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 (3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm.
  • the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6.0, conductivity 15 mS/cm.
  • the column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6.0 buffer, at a conductivity of 30 mS/cm.
  • Impurities bound to the column were subsequently washed with 3 CV washing with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 at a conductivity 48 mS/cm.
  • Example 2 the eluate from Example 1 may be subjected to cation exchange chromatography.
  • Eluate from Example 1 is concentrated and the conductivity and pH are reduced by diafiltration by a TFF step using 73 mM sodium acetate buffer, pH 4.8.
  • This step acts as a buffer exchanging step wherein the pooled eluate of Q-Sepharose column is brought into 73 mM sodium acetate buffer of pH 4.8 conductivity 4 mS/cm.
  • the buffer exchanged sample is then loaded onto a CM-Sepharose column (300 ml_, XK 50/20) that was pre- equilibrated with 73 mM sodium acetate buffer, pH 4.8.
  • the flow through fraction from Example 3 containing the desired protein is concentrated and exchanged with buffer containing 83.4 mM sodium acetate (or 80-85 mM sodium acetate) pH 3.3 (or pH 3-3.5) using TFF.
  • Sample from the TFF is loaded onto a MEP chromatographic column (4 ml_, Tricorn 5/20) pre- equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 (3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm.
  • the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6, conductivity 15 mS/cm.
  • the column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6, at a conductivity of 30 mS/cm.
  • Impurities bound to the column are subsequently washed with 3 CV wash with 25 mM Tris, 500 mM NaCI buffer of pH 7.1 at a conductivity of 48 mS/cm.
  • the eluate fractions from hydrophobic anion induction chromatography of Example 4 are analyzed by isoelectric focusing (IEF).
  • the IEF gel is prepared using water, urea, 30% acrylamide, and Ampholyte (pH range 2-4 and 3-10). The above components are mixed gently and 10% w/v ammonium persulfate and TEMED are added to the mixture and the mixture is cast in a gel sandwich apparatus (BIORAD Mini Protean Cell) and fitted with a comb. The gel is allowed to polymerize for 45 minutes at room temperature. A small amount of protein solution (sample) is mixed with an equal volume of sample buffer (glycerol, Ampholyte and Milli-Q water) and protein samples are loaded into the gel.
  • sample buffer glycerol, Ampholyte and Milli-Q water
  • the gel is then placed in a BIORAD Mini Protean Cell assembly and filled with a cathode buffer (25 mM sodium hydroxide) and anode buffer (25 mM orthophosphoric acid) in separate compartments.
  • a cathode buffer 25 mM sodium hydroxide
  • anode buffer 25 mM orthophosphoric acid
  • the isoelectric focusing procedure can also be used with eluate fractions from hydrophobic anion induction chromatography of Examples 2 and 6.
  • Fig. 1 is an illustration of a chromatogram from the procedure as described in this example.
  • the line marked “Cond” shows the increase in conductivity in mS/cm. Peaks A and B represent the eluate obtained at conductivities 15 mS/cm and 30 mS/cm, respectively.
  • Fig. 2 is an isoelectric focusing gel of the hydrophobic anion induction chromatography performed as described in this example. Lane 1 is the internal reference standard. Lane 2 corresponds to fraction obtained by elution at pH 6 and conductivity 15 mS/cm. Lane 3 corresponds to fractions obtained by elution at pH 6 and conductivities 30 mS/cm.
  • Fig. 3 shows a comparison of darbepoetin alpha samples from different batches. Lanes 1 -9 corresponds to darbepoetin alpha samples from different batches. Lane 10 corresponds to a commercially available darbepoetin alpha product, Aranesp® (pi 3-3.9). Lane 3 is used as an internal reference standard.
  • cell culture broth from the Expression and Harvest of Protein procedure above may be subjected to an affinity chromatography procedure.
  • the cell culture broth is loaded onto a Blue-Sepharose FF (20 mL, VL 1 1 /21 ) column that is pre-equilibrated with 5 CV of 50 mM phosphate, 100 mM NaCI, pH 7.5 buffer at a conductivity of 8 mS/cm.
  • the desired protein bound onto the column is eluted using 250 mM Tris buffer of pH 7.5.
  • a phenylboronate column (20 mL, VL 1 1/21 ) may be used as an affinity chromatography step wherein the crude extract of the clarified cell culture broth is loaded onto the phenylboronate column.
  • the column is pre- equilibrated with 5 CV of 50 mM phosphate, 100 mM NaCI, pH 7.5 buffer at a conductivity of 8 mS/cm.
  • the desired protein bound onto the column is eluted using 50 mM phosphate, 100 mM NaCI, 100 mM sorbitol buffer of pH 7.5.
  • Sample from TFF is loaded onto a MEP chromatographic column (4 mL, Tricorn 5/20) pre-equilibrated with 5 CV of 83.4 mM sodium acetate buffer of pH 3.3. After loading, the column is washed with 83.4 mM sodium acetate buffer of pH 3.3 ( 3 CV) followed by a wash of 73 mM acetate buffer, pH 4.8, conductivity 4 mS/cm.
  • the column is treated with 10 CV of 20 mM phosphate, 140 mM NaCI, pH 6, conductivity 15 mS/cm.
  • the column is further washed with 3 CV of 40 mM phosphate, 280 mM NaCI, pH 6, conductivity 30 mS/cm.
  • the column is subsequently washed with 3 CV of 25 mM Tris, 500 mM NaCI buffer of pH 7.1 and conductivity 48 mS/cm.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention porte sur des procédés efficaces de purification d'une cytokine modifiée. Les procédés consistent à utiliser une technique chromatographique pour purifier la cytokine désirée. La cytokine purifiée peut être utilisée pour préparer des compositions thérapeutiques.
PCT/US2011/039439 2010-06-07 2011-06-07 Purification de cytokines modifiées WO2011156369A2 (fr)

Applications Claiming Priority (2)

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IN1567/CHE/2010 2010-06-07
IN1567CH2010 2010-06-07

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WO2011156369A2 true WO2011156369A2 (fr) 2011-12-15
WO2011156369A3 WO2011156369A3 (fr) 2012-04-05

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140171626A1 (en) * 2012-12-19 2014-06-19 Glaxosmithkline Llc Protein purification
WO2014204023A1 (fr) * 2013-06-17 2014-12-24 씨제이헬스케어 주식회사 Nouveau procédé de purification pour la darbépoétine alpha
US20170022257A1 (en) * 2013-11-29 2017-01-26 Cj Healthcare Corporation Method for purifying darbepoetin alfa
EP3428284A4 (fr) * 2016-03-09 2019-09-04 JCR Pharmaceuticals CO., LTD. Procédé de production d'érythropoïétine humaine mutante

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5641870A (en) * 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US20030023043A1 (en) * 2000-03-02 2003-01-30 Kazuhisa Uchida Method of separating and purifying protein
WO2001064711A1 (fr) * 2000-03-02 2001-09-07 Kyowa Hakko Kogyo Co., Ltd. Procede de separation et de purification de proteine
RS50516B (sr) * 2003-11-05 2010-05-07 Ares Trading S.A. Prečišćavanje il-18 vezujućeg proteina
US20080071067A1 (en) * 2006-09-20 2008-03-20 Avigenics, Inc. Methods of purifying proteins from egg white

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140171626A1 (en) * 2012-12-19 2014-06-19 Glaxosmithkline Llc Protein purification
WO2014204023A1 (fr) * 2013-06-17 2014-12-24 씨제이헬스케어 주식회사 Nouveau procédé de purification pour la darbépoétine alpha
US20170022257A1 (en) * 2013-11-29 2017-01-26 Cj Healthcare Corporation Method for purifying darbepoetin alfa
US10723775B2 (en) * 2013-11-29 2020-07-28 Cj Healthcare Corporation Method for purifying darbepoetin alfa
EP3428284A4 (fr) * 2016-03-09 2019-09-04 JCR Pharmaceuticals CO., LTD. Procédé de production d'érythropoïétine humaine mutante
US10604779B2 (en) 2016-03-09 2020-03-31 Jcr Pharmaceuticals Co., Ltd. Method for production of mutant-type human erythropoietin

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