WO2019077432A1 - Procédé de purification amélioré de pth (1-34) de recombinaison - Google Patents

Procédé de purification amélioré de pth (1-34) de recombinaison Download PDF

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Publication number
WO2019077432A1
WO2019077432A1 PCT/IB2018/057709 IB2018057709W WO2019077432A1 WO 2019077432 A1 WO2019077432 A1 WO 2019077432A1 IB 2018057709 W IB2018057709 W IB 2018057709W WO 2019077432 A1 WO2019077432 A1 WO 2019077432A1
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WIPO (PCT)
Prior art keywords
diafiltration
ultrafiltration
output
pth
protein
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PCT/IB2018/057709
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English (en)
Inventor
Sridevi KHAMBHAMPATY
Mili Pathak
Irshad GANDHI
Parag GAJJAR
Janani PALANISAMY
Ronak MALDE
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Intas Pharmaceuticals Ltd.
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Publication of WO2019077432A1 publication Critical patent/WO2019077432A1/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
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis

Definitions

  • the present invention relates to improved method for purification of recombinant PTH (1-34) with decreased protein precipitation or particle formation.
  • Parathyroid hormone is a polypeptide hormone which is naturally produced in the parathyroid of mammals.
  • the human polypeptide consists of 84 amino acids and is involved in the regulation of the calcium concentration in blood plasma. If the calcium level drops below a threshold level, PTH is secreted by cells of the parathyroid glands into the blood and induces a release of calcium from bone tissue. At the same time, PTH supports calcium absorption from the small intestine and enhances calcium reabsorption from the primary urine, thereby suppressing calcium loss via the kidneys.
  • PTH (1- 34) is a polypeptide having a molecular mass of 4.7 kDa that consists of the first 34 amino acids of the human PTH hormone. It was approved for osteoporosis therapy in 2002 under the product name "Teriparatide”. Teriparatide is sold by Lilly Pharma under the trade name Forteo (in US) and Forsteo (in Europe). It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).
  • IBs insoluble inclusion bodies
  • Purification of protein is a series of processes intended to isolate a single type of protein from a complex mixture, besides being essential for the characterization of its structure and function.
  • the starting material is usually a biological tissue or a microbial culture expressing PTH (1-34).
  • the various steps in the purification process may free the protein from a matrix that confines it, separate the protein and nonprotein parts of the mixture, and finally separate the desired protein from all other proteins and impurities. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity to variety of chromatographic resins under different conditions.
  • recombinant teriparatide Because of the importance of recombinant teriparatide in the treatment of osteoporosis, the provision of recombinant teriparatide of high purity and high specific activity is desirable. Recombinant teriparatide requires repeated injections. Highly purified recombinant teriparatide preparations can be administered subcutaneously, permitting self-administration by the patient and thereby increasing patient convenience and compliance.
  • PTH for therapeutic use is produced by heterologous expression in bacterial host cells and subsequent purification of the pharmacological active polypeptide. It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain.
  • the PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).
  • IBs insoluble inclusion bodies
  • the fusion protein is treated enzymatically with the enzyme corresponding to the selective cleavage site.
  • the fusion protein in its more impure state, even in refractile form can be treated with the enzyme. If needed, the resulting mature PTH, or variant thereof, can be further purified.
  • Escherichia coli expression system serves to be the most popular and widely used expression system for production of recombinant therapeutic protein in form of inclusion bodies (IBs).
  • IBs inclusion bodies
  • These IBs are dense, with tightly packed aggregated proteins and this aggregated protein are converted into fully active native form by refolding process.
  • Refolding in batch process using drip dilution (of solubilized and reduced IBs) method is the most preferred and widely used.
  • the refolding buffer contains redox components, buffering agent, chelating agents and stabilizers etc.
  • Post refolding includes several downstream unit operations to purify the recombinant therapeutic proteins. Due to volume handling issues when operated at large scale, the refolding output is concentrated and buffer exchanged to condition it for the next chromatography step.
  • arginine as the most preferred and extensively used additive in refolding of recombinant proteins. Arginine is widely used to suppress protein aggregation. Arginine reduces attractive protein- protein interactions, and increases the solubility of unfolded species thereby decreasing the rate of association of unfolded and partially folded intermediates on the folding pathway during refolding.
  • WO2008031020 discloses methods for isolating a product and/or reducing turbidity and/or impurities from a load fluid comprising the product and one or more impurities by passing the load fluid through a medium, followed by at least one wash solution comprising arginine or an arginine derivative and collecting the product using an elution solution.
  • US 6103495 describes the purification process comprising the following steps (a) separating host cells from the culture medium; (b) subjecting the medium to reverse-phase liquid chromatography and recovering fractions containing peptide product; (c) subjecting said fractions of step (b) to cation exchange chromatography and (d) thereafter recovering fractions containing peptide product.
  • US6210925 describes the purification process comprising the following steps: (a) separating host cells from the culture medium; (b) subjecting the medium to cation exchange chromatography and recovering fractions containing said peptide product; (c) subjecting the recovered fraction of step (b) to reverse-phase liquid chromatography and recovering fractions containing peptide product; (d) subjecting the recovered fractions of step (c) to cation exchange chromatography and (e) thereafter recovering fractions containing peptide product.
  • WO2009129226 discloses method for concentrating a protein, in particular a method for concentrating a plasma product, in particular IgG, using glycine in a two-stage ultrafiltration/diafiltration approach.
  • WO2009019715 discloses two steps orthogonal purification process for recombinant human PTH (1-34) comprising of cation exchange chromatography optionally followed by preparative chromatography selected from HIC or RP-HPLC to yield a target protein of > 99%.
  • WO2015025335 discloses process for purification of parathyroid hormone using enzymatic cleavage; anion exchange chromatography followed by other suitable purification step.
  • US20170129935 discloses method for purifying PTH (1-34) by cation exchange chromatography.
  • Tripathi ChemBioEng Reviews, 3: 116-133, 2016 discloses refolding process of recombinant proteins produced in E.coli and purification using various chromatographic steps.
  • present invention uses critical arginine concentration in the concentrated refolding output solution.
  • the improved method of the present invention provides for the effective purification of the recombinant PTH (1-34) polypeptide by ultrafiltration diafiltration (UFDF) followed by subsequent purification steps. Accordingly, it would be desirable to provide a method for purifying recombinant PTH (1-34) by decreasing protein aggregation/precipitation and particle formation along with increase in optical density to achieve higher final concentrations along with minimizing yield loss and impact on processing time.
  • the principal object of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another object of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another object of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another object of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps.
  • Another object of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another object of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:
  • Another object of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:
  • the principal aspect of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps.
  • Another aspect of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another aspect of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • Another aspect of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration three times with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps.
  • Another aspect of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another aspect of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:
  • Another aspect of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of: (a) Solubilization of Inclusion bodies (IBs) of recombinant PTH (1-34);
  • Figure-1 represents decrease in turbidity of recombinant PTH (1-34) due to reduction in precipitation / particle formation (aggregate) and simultaneous increase in its optical density (OD).
  • Recombinant human parathyroid hormone PTH (1-34) is a biologically active N-terminal fragment of endogenous human parathyroid hormone (PTH).
  • PTH a biologically active N-terminal fragment of endogenous human parathyroid hormone (PTH).
  • Therapeutically, PTH (1-34) is used for the treatment of men and postmenopausal women with osteoporosis who are at high risk of fracture. It increases bone mineral density and reduces the risk of vertebral and non-vertebral fractures.
  • the Parathyroid hormone is a protein of 84 amino acid residues, of which the first 34 amino acids (1-34) are necessary for biological activity.
  • Recombinant human parathyroid hormone (PTH) is a 4117.8 Daltons polypeptide chain consisting of the first 34 amino acids of the parathyroid hormone, with a pi of 8.69. It is produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain. The PTH fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).
  • Inclusion bodies have higher density (-1.3 mg ml -1 ) than many of the cellular components, and thus can be easily separated by high-speed centrifugation after cell disruption.
  • Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. Aggregation is the leading cause of decreased refolding yields.
  • the IBs are released by cell lysis with a high pressure homogenizer and recovered by centrifugation. Several washing and centrifugation steps are performed to wash the IBs off cellular debris, before solubilization. The solubilized IBs are then subjected to refolding following which the fusion partner is separated by proteolysis and PTH ( 1 -34) is purified through a series of chromatographic unit operations.
  • Inclusion bodies are dense electron-refractile particles of aggregated protein found in both the cytoplasmic and periplasmic spaces of E. coli during high-level expression of heterologous protein. It is generally assumed that high level expression of non-native protein (higher than 2% of cellular protein) and highly hydrophobic protein is more prone to lead to accumulation as inclusion bodies in E. coli. In the case of proteins having disulfide bonds, formation of protein aggregates as inclusion bodies is anticipated since the reducing environment of bacterial cytosol inhibits the formation of disulfide bonds.
  • the downstream process for purifying fusion protein involves solubilization of inclusion bodies, refolding, ultrafiltration diafiltration, digestion and chromatography steps for preparation of drug substance.
  • refolding output is concentrated and buffer exchanged in diafiltration buffer at room temperature.
  • Post ultrafiltration diafiltration the ion exchange chromatography followed by the digestion of the fusion protein by enzyme is carried out.
  • MMC mixed mode chromatography
  • the successive step of ion exchange chromatography removes the truncated impurities.
  • Gel filtration chromatography is performed to buffer exchange in the formulation buffer.
  • the main embodiment of the present invention is to provide an improved process for purification of protein with decreased protein precipitation or particle formation through ultrafiltration diafiltration (UFDF) followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another embodiment of the present invention is to provide an improved process for purifying a protein through ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another embodiment of the present invention is to provide highly efficient scalable process for purification of recombinant PTH; comprising a step of diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation and simultaneously increase in optical density followed by subsequent purification steps.
  • Another embodiment of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another embodiment of the present invention is to provide an improved process for purifying recombinant PTH (1-34) by ultrafiltration diafiltration (UFDF) in which arginine concentration is maintained by diluting output of ultrafiltration with diafiltration buffer before processing through diafiltration in order to decrease precipitation or particle formation, optionally final diafiltered output can be again concentrated in the continuous manner in same setup to reduce volume handling followed by subsequent purification steps.
  • UFDF ultrafiltration diafiltration
  • Another embodiment of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:
  • Another embodiment of the present invention to provide an improved process for purifying recombinant PTH (1-34), wherein said method comprising the steps of:
  • the ultrafiltration output obtained as per the present invention is diluted more than one time with diafiltration buffer before processing through diafiltration; preferably three times.
  • method of present invention results in reduction of protein precipitation or particle formation by ⁇ 7 folds. Due to use of present invention during UFDF, the output of subsequent chromatography step increases by 20-30% thereby increasing the yield.
  • the present invention is an efficient and scalable process for purification of recombinant PTH (1-34) as by following present invention there is increase of more than 20% productivity after MMC output.
  • Recombinant PTH (1-34) is used as a model protein to demonstrate present invention.
  • rPTH (1- 34) was produced in the form of a fusion protein by recombinant DNA technology using a genetically engineered E. coli strain.
  • the rPTH (1-34) fusion protein expressed resides in the cytoplasm of E. coli in the form of insoluble inclusion bodies (IBs).
  • IBs insoluble inclusion bodies
  • the IBs were released by cell lysis with a high pressure homogenizer and recovered by centrifugation. Several washing and centrifugation steps were performed to wash the IBs off cellular debris, before solubilization. The solubilized IBs were then subjected to refolding.
  • Ultrafiltration Diafiltration output was further subjected to cation exchange chromatography - I (CEX-I) followed by enzymatic digestion through use of Enterokinase, mixed mode chromatography (MMC), cation exchange chromatography - II (CEX-II) and gel filtration chromatography (GFC).
  • MMC mixed mode chromatography
  • CEX-II cation exchange chromatography - II
  • GFC gel filtration chromatography
  • Table 3 Average total optical densities obtained by present invention
  • the increase in productivity at CEX-I output and at MMC output was respectively 31.31% and 23.08% represents the effectiveness of highly scalable present purification process of rPTH (1- 34) in comparison with traditional method.
  • the comparison of increase in productivity was done at CEX-I output and MMC output step as CEX-I was immediate step after UFDF 1 and purified the fusion protein.
  • MMC purified the cleaved PTH from the fusion protein.

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  • Health & Medical Sciences (AREA)
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  • Endocrinology (AREA)
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  • Peptides Or Proteins (AREA)
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Abstract

La présente invention concerne un procédé amélioré de purification de PTH (1-34) de recombinaison avec une précipitation de protéines ou une formation de particules réduite.
PCT/IB2018/057709 2017-10-16 2018-10-04 Procédé de purification amélioré de pth (1-34) de recombinaison WO2019077432A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113480622A (zh) * 2021-08-05 2021-10-08 江苏坤力生物制药有限责任公司 一种重组肺炎球菌溶血素制备及纯化的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195888A1 (en) * 2007-11-30 2013-08-01 Abbvie Ultrafiltration and diafiltration formulation methods for protein processing
WO2015025335A1 (fr) * 2013-08-21 2015-02-26 Cadila Healthcare Limited Procédé de purification de pth
EP2857417A1 (fr) * 2012-06-05 2015-04-08 CJ Healthcare Corporation Protéine d'hormone de croissance humaine à longue action hautement glycosylée, et son procédé de production

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130195888A1 (en) * 2007-11-30 2013-08-01 Abbvie Ultrafiltration and diafiltration formulation methods for protein processing
EP2857417A1 (fr) * 2012-06-05 2015-04-08 CJ Healthcare Corporation Protéine d'hormone de croissance humaine à longue action hautement glycosylée, et son procédé de production
WO2015025335A1 (fr) * 2013-08-21 2015-02-26 Cadila Healthcare Limited Procédé de purification de pth

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113480622A (zh) * 2021-08-05 2021-10-08 江苏坤力生物制药有限责任公司 一种重组肺炎球菌溶血素制备及纯化的方法

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