WO2004048588A1 - Procede pour purifier un produit de fermentation - Google Patents

Procede pour purifier un produit de fermentation Download PDF

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Publication number
WO2004048588A1
WO2004048588A1 PCT/DK2003/000801 DK0300801W WO2004048588A1 WO 2004048588 A1 WO2004048588 A1 WO 2004048588A1 DK 0300801 W DK0300801 W DK 0300801W WO 2004048588 A1 WO2004048588 A1 WO 2004048588A1
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WO
WIPO (PCT)
Prior art keywords
fermentation
process according
less
derived product
fermentation broth
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Application number
PCT/DK2003/000801
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English (en)
Inventor
Jan Markussen
Ivan Diers
Original Assignee
Novo Nordisk A/S
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 Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to EP03773588A priority Critical patent/EP1567657A1/fr
Priority to AU2003281982A priority patent/AU2003281982A1/en
Publication of WO2004048588A1 publication Critical patent/WO2004048588A1/fr

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Classifications

    • 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
    • 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
    • C07K14/605—Glucagons
    • 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
    • C07K14/62—Insulins

Definitions

  • the present invention relates to a simple process for purification of fermentation-derived products. More specifically the processes of the invention pertain to heat treatment of culture broth for precipitation and removal of impurities.
  • the conventional method for recovering fermentation-derived products, such as proteins and antibiotics, from the complex culture broth matrix is commonly liquid chromatography.
  • This process comprises the application of the product holding fluid onto a solid chromatographic matrix under conditions where the fermentation-derived product binds to the chromatographic matrix while the bulk of impurities pass through the chromatographic column. After a washing phase the bound product is eluted from the column.
  • the method eliminates the major part of host cell impurities from the product.
  • chromatography is an expensive method for recovery of fermentation derived products.
  • chromatography is not well suited for continuous processes which are often used in the industrial manufacture of fermentation- derived products.
  • chromatographic column operation is not robust towards normal Krutation-denved impurities such as remnant cells and cellular debris, anfifoam, host cells proteins and proteases. Often many sequential steps are needed for a chromatographic recovery, including upstream centrifugation and filtration steps and several chromatographic steps each targetting a certain group of impurities.
  • Membrane filtration such as microfiltration and ultrafiltration has also been used for the purification steps following fermentation with some success.
  • membrane filtration processes are often quite slow and relatively expensive processes.
  • Addition of flocculation agents have also been applied as the initial purification step for pro- teins (WO 96/38469 and Biotechnol. Prog. 16, 2000, 661-667), but it is expensive and gives rise to waste disposal problems.
  • fermentation-derived products such as protein and antibiotics should be kept in solution at as low temperatures as possible in order to prevent microbial, enzymatic or chemical degradation of the product (Biochemical engineering fundamentals, J.E. Bailey, D.F. Ollis, McGraw-Hill Inc., 1986).
  • the present invention provides a method for the industrial manufacture of fermentation- derived products, which enables continuous manufacturing and better separation of product and impurities while reducing manufacturing costs and reducing down-time of chromatographic columns.
  • Fermentation derived products or precursors thereof are commonly produced by cultivation of recombinant host cells, e.g. bacteria, fungi and mammalian cells, in an appropriate fer- mentation medium.
  • the fermentation medium may be chemically defined or it may be a complex medium containing the necessary nutrients for growth and product formation of the host cells, e.g. sugar, nitrogen source, salts, vitamins etc.
  • the fermentation broth contains the desired product in a mixture with remnant medium components and host cell derived impurities.
  • Host cell derived impurities are mainly proteins, nucleic acids and, in particular where an intracellular product is released by disrupting the cells, cellular debris.
  • the first step in the recovery or purification of the fermentation derived product is to separate the major part of the host cell derived impurities from the product and to concentrate the product.
  • the present invention relates to a process for purifying a fermentation-derived product, said process comprising the steps of : a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product.
  • purifying a fermentation-derived product means the separation of the fermentation-derived product from impurities present in the starting material. Thus, the separation results in the fermentation-derived product being of higher purity than that in the starting material.
  • fermentation-derived product means the product compound being produced by the overall manufacturing process.
  • the fermentation-derived product may be a compound which is directly synthesised by the host cells, or it may be a chemical derivative or fragment of a precursor produced by the host cells.
  • Chemical derivatives can be esters, acylated forms and PEGylated molecules.
  • the term "precursor” as used herein means a covalently modified form which can be converted into the desired form.
  • the fermentation-derived product may either be the protein itself or more often a precursor thereof.
  • the precursor typically is the product protein with an amino acid extension which increases the yield in the fermentation process or which facilitates purification steps such as affinity chromatography, e.g. IMAC purification of his-tagged proteins.
  • the precursor can also be the parent protein when the fermentation-derived product is a chemically modified form of the protein.
  • fermentation broth as used herein means the product holding fluid which results from the fermentation process.
  • the term “fermentation broth” encompasses solutions and suspensions, i.e. the cell free supernatant, the broth with whole cells and the broth with or without cellular debris following cell disruption as well as broth resulting from any solubihsa- tion steps or protein refolding steps.
  • the present invention relates to a process for purifying a fermentation- derived product, said process comprising the steps of : ' a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product; wherein no flocculation agent is added to said fermentation broth.
  • flocculation agent means chemicals which are added to the fermentation broth after the fermentation has stopped in order to bind impurities forming insoluble complexes which subsequently precipitates.
  • flocculation agents are Fe 2+ , Al 3+ and a range of charged polymers.
  • the soluble portion of the fermentation broth in step c) contains at least 60% of the product which results in the fermentation derived product.
  • the pH of the fermentation broth which is heated in step a) is at least 1 pH unit, preferable at least 2 pH units from the isoelectric point of said fermentation-derived product.
  • the mean residence time of the fermentation broth at temperatures in the range from 60 °C to 90 °C in step a) is less than 60 minutes, less than 30 minutes, less than 15 minutes, most preferable less than 10 minutes.
  • the fermentation broth is cooled to temperatures below 35 °C in step b).
  • the temperature of the fermentation broth during the separation step c) is less than 40 °C, less than 35 °C, less than 25 °C or less than 10 °C.
  • the separation in step c) is performed by centrifugation.
  • Large scale centrifuges for industrial applications are commercially available.
  • Preferred centrifuges are for continuous operation, e.g. solids ejecting centrifuges and decanter centrifuges.
  • the separation in step c) is performed by microfiltration.
  • a number of industrial scale microfiltration units are available for cross-flow microfiltration or vibrating microfiltration.
  • Microfiltration membranes may be formed from a variety of materials such as natural polymers, synthetic polymers, ceramics and metals.
  • Preferred microfiltration membranes are ceramic membranes which may be formed by fibres of silicon carbide, silicon nitride, aluminosilicate, mixtures thereof and which may optionally be carbon-coated (see e.g. WO 00/45938).
  • Preferred metal microfiltration membranes are zirconium membranes.
  • the nominal pore size of MF membranes are typically in the range from 0.01 ⁇ m to 100 ⁇ m, preferably from 0.05 ⁇ m to 75 ⁇ m and more preferable from 0.1 ⁇ m to 50 ⁇ m.
  • the MF process is typically carried out using cross flow filtration where the broth also flows along the membrane surface.
  • the process steps a), b) and c) are run in continuous mode.
  • the present invention relates to a process for purifying a fermentation- derived product, said process comprising the steps of : a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating of the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product; wherein said soluble portion of the fermentation broth produced in step c) is subjected to col- umn chromatography.
  • the present invention relates to a process for purifying a fermentation- derived product, said process comprising the steps of : a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product; wherein said soluble portion of the fermentation broth produced in step c) is subjected to crystallization or precipitation.
  • the present invention relates to a process for purifying a fermentation- derived product, said process comprising the steps of : a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product; wherein said soluble portion of the fermentation broth produced in step c) is subjected to ul- trafiltration.
  • the cut-off value of the UF membrane is lower than four times the molecular weight of the fermentation- derived product, preferably lower than twice the molecular weight of the fermentation-derived product and most preferably lower than the molecular weight of the fermentation-derived product.
  • the product holding fluid resulting from said ultrafiltration is subjected to column chromatography.
  • the present invention relates to a process for purifying a fermentation- derived product, said process comprising the steps of : a) heating the fermentation broth containing said fermentation-derived product or a precursor thereof to a temperature in the range from 60 °C to 90 °C, b) cooling the fermentation broth to a temperature below 60 °C; c) separating the precipitate from the soluble portion of the fermentation broth at a temperature less than 60 °C; d) isolating said fermentation-derived product; wherein said fermentation-derived product is a protein.
  • said fermentation-derived product is a pharmaceutical protein or a precursor thereof.
  • pharmaceutical protein as used herein means a protein which has a known pharmaceutical activity.
  • said fer- mentation-derived product is a commercialised pharmaceutical protein.
  • commercialised pharmaceutical protein means a pharmaceutical protein which has been approved by a regulatory agency in at least one country selected from US and EU countries.
  • said fermentation-derived product is produced by a recombinant host cell.
  • said host cells are selected from the group consisting of Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Pichia methanolica, Candida utilis and Kl ⁇ yveromyces lactis.
  • said fermentation-derived product or a precursor thereof has a molar weight of less than 25000 Dalton, less than 10000 Dalton, less than 7000 Dalton, or less than 4000 Dalton.
  • said pro- tein is selected from the group consisting of GLP-1 , exendin-4, exendin-3, GLP-2, glucagon, TFF peptides, interleukins, insulin, albumin, precursors thereof and analogs of any of the foregoing.
  • said protein is Ser 38 ,Lys 39 ' 40 ' 41 - 42, 43 ' 44 -Exendin-4(1-39)-amide (ZP-10).
  • analog as used herein means a variant of a protein wherein one or more amino acid residues of the parent protein has been substituted by other amino acid residue(s) and/or wherein one or more amino acid residues have been inserted into the parent protein and/or wherein one or more amino acid residues have been deleted from the parent protein.
  • an analog differs from the parent protein in no more than five amino acid residues.
  • an analog differs from the parent peptide in no more than three amino acid residues.
  • an analog differs from the parent peptide in only one amino acid residue.
  • said protein is selected from the group consisting of human insulin, a human insulin precursor, a hu- man insulin analog, a human insulin analog precursor, Arg 34 -GLP-1(7-37) and GluGluAlaGluLys-Arg 34 -GLP-1(7-37).
  • the peptide SCI-13 has the sequence: (B-chain)-Gly-Tyr-Gly-Asn-His-Asp-Leu-Asn-Phe-Pro- Gln-Thr-(A-chain), wherein (B-chain) is the 30 amino acid B-chain of human insulin, and (A- chain) is the 21 amino acid A-chain of human insulin. SCI-13 thus has a 12 amino acid pep- tide connecting the C-terminus of the B-chain to the N-terminus of the A-chain.
  • a 4 x 150 mm column of C-185 ⁇ Licrosorb was used and the effluent analysed by UV- detection at 214 nm.
  • a linear gradient from 90% buffer A (0.018 M (NH) 4 SO , 0.0125 M Tris, 20% CH 3 CN, pH 7.0) and 10% B (50% CH 3 CN) to 20% buffer A and 80% B was applied during 20 minutes using a pumping rate of 1.5 ml/min.
  • a standard of human insulin emerges in this system at 12.8 min and the SCI-13 compound emerges at 12.1 min.
  • the results of the experiment shows that impurities are precipitated and that the SCI-13 compound is rendered fully soluble by the heat treatment of the broth.
  • the solution is conditioned for further purification steps by column chromatography or other processes where it is desirable that the product is in freely soluble form.
  • Clarification of supernatant by heat treatment before preparative chromatography Clarification of supernatant by heat treatment before preparative chromatography.
  • Fermentation broth from yeast strain YES2507 expressing Arg ⁇ -GLP-I (7-37) with the N- terminal extension GluGluAlaGluLys (EEAEK) was prepared by fermentation as described in Example 1.
  • the GLP-1 analog was solubilised and cells were removed by centrifugation after adjustment of the 4.2 litres of broth to pH 9.7 by adding NaOH, and pH was then quickly adjusted to 3.0 in the supernatant (3.5 litres) by addition of hydrochloric acid.
  • the unclear and brown coloured liquid was subjected to heat treatment in a 10 litre fermentor equipped with a heating/cooling jacket. Temperature was raised from ambient to 80°C in 3-4 minutes by injection of steam into the jacket and slow stirring of the liquid for heat transfer.
  • the tempera- ture was kept constant at 80°C for 10 minutes and subsequently cooled quickly to ambient temperature by circulation of 5°C cooling water in the jacket. The dark coloured precipitate was removed by centrifugation to give a final clear, light brown solution of 3.25 litres. This clear solution was then directly applied to a chromatography column with no further treatment. The concentration of Arg ⁇ -GLP-I (7-37) in the clear solution was determined by HPLC as described in Example 1.
  • Broth from a yeast fermentation producing GluGluAlaGluLys-Arg 34 -GLP-1(7-37) is collected and stored below 10°C prior to recovery.
  • the fermentation broth was then clarified for yeast cells by means of centrifugation.
  • the resulting supernatant has a pH of 5.8 and a turbidity of 35 NTU units (Nephelometric Turbidity Unit).
  • the supernatant pH is then adjusted to 3.0 by addition of sulfuric acid whereby the turbidity increases to 76 NTU.
  • One part of the acidified supernatant is then heat treated at 80°C for 10 minutes by passing the liquid through an heat exchanger unit using a mean residence time of 10 minutes. The heated liquid is cooled to below 10°C once it leaves the heat exchanger.
  • the second half of the supernatant is considered reference material and stored below 10°C. Both the heat treated supernatant and the reference material are centrifuged and the super- natants from these centrifugations are collected. The turbidity of both ice cooled super- natants was measured to:
  • Turbidity reference material 76 NTU

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
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Abstract

L'invention concerne un procédé pour purifier un produit de fermentation, ce procédé comprenant les étapes suivantes: a) chauffer le bouillon de fermentation contenant le produit de fermentation ou un précurseur de celui-ci à une température d'au moins 60 °C; b) refroidir le bouillon de fermentation à une température inférieure à 60 °C; c) séparer le précipité de la partie soluble du bouillon de fermentation à une température inférieure à 60 °C; d) isoler ledit produit de fermentation.
PCT/DK2003/000801 2002-11-26 2003-11-24 Procede pour purifier un produit de fermentation WO2004048588A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP03773588A EP1567657A1 (fr) 2002-11-26 2003-11-24 Procede pour purifier un produit de fermentation
AU2003281982A AU2003281982A1 (en) 2002-11-26 2003-11-24 Process for purifying a fermentation-derived product

Applications Claiming Priority (4)

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DKPA200201821 2002-11-26
DKPA200201821 2002-11-26
US43074802P 2002-12-04 2002-12-04
US60/430,748 2002-12-04

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006101441A2 (fr) * 2005-03-24 2006-09-28 Straumann Holding Ag Nouvelle methode de purification de proteines
WO2015166037A1 (fr) * 2014-04-30 2015-11-05 Novozymes A/S Procédé de réduction du taux d'adn d'un bouillon de fermentation
CN105111306A (zh) * 2015-08-28 2015-12-02 北京工业大学 美洲短吻鳄白蛋白的分离方法
WO2019223752A1 (fr) * 2018-05-24 2019-11-28 江苏恒瑞医药股份有限公司 Procédé de préparation d'un précurseur d'insuline humaine recombinée ou d'un analogue associé
WO2022130324A1 (fr) * 2020-12-18 2022-06-23 Glycom A/S Élimination de biomasse par centrifugation

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Publication number Priority date Publication date Assignee Title
WO1990000200A1 (fr) * 1988-06-27 1990-01-11 Genex Corporation Liberation thermique d'une proteine recombinante dans un milieu de culture
EP0431679A1 (fr) * 1989-12-05 1991-06-12 Merck & Co. Inc. Procédé de stabilisation de protéines recombinantes de surface du virus de l'hépatitis B à partir de levure
EP0438767A2 (fr) * 1990-01-25 1991-07-31 BASF Aktiengesellschaft Procédé de séparation de riboflavine d'une suspension de fermentation
US5455331A (en) * 1987-05-14 1995-10-03 Commonwealth Scientific And Industrial Research Organisation Enriched whey protein fractions and method for the production thereof
EP0699687A2 (fr) * 1994-08-31 1996-03-06 The Green Cross Corporation Procédé de purification du sérum albumine humaine
JPH10101696A (ja) * 1996-08-08 1998-04-21 Shinotesuto:Kk 形質転換体にて発現される蛋白質に含まれる夾雑物質の除去方法及び精製蛋白質
EP1329462A1 (fr) * 2000-10-24 2003-07-23 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Procede d'elaboration d'albumine de serum d'origine humaine comportant une etape de chauffage

Patent Citations (7)

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Publication number Priority date Publication date Assignee Title
US5455331A (en) * 1987-05-14 1995-10-03 Commonwealth Scientific And Industrial Research Organisation Enriched whey protein fractions and method for the production thereof
WO1990000200A1 (fr) * 1988-06-27 1990-01-11 Genex Corporation Liberation thermique d'une proteine recombinante dans un milieu de culture
EP0431679A1 (fr) * 1989-12-05 1991-06-12 Merck & Co. Inc. Procédé de stabilisation de protéines recombinantes de surface du virus de l'hépatitis B à partir de levure
EP0438767A2 (fr) * 1990-01-25 1991-07-31 BASF Aktiengesellschaft Procédé de séparation de riboflavine d'une suspension de fermentation
EP0699687A2 (fr) * 1994-08-31 1996-03-06 The Green Cross Corporation Procédé de purification du sérum albumine humaine
JPH10101696A (ja) * 1996-08-08 1998-04-21 Shinotesuto:Kk 形質転換体にて発現される蛋白質に含まれる夾雑物質の除去方法及び精製蛋白質
EP1329462A1 (fr) * 2000-10-24 2003-07-23 Juridical Foundation, The Chemo-Sero-Therapeutic Research Institute Procede d'elaboration d'albumine de serum d'origine humaine comportant une etape de chauffage

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* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 1998, no. 09 31 July 1998 (1998-07-31) *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006101441A2 (fr) * 2005-03-24 2006-09-28 Straumann Holding Ag Nouvelle methode de purification de proteines
WO2006101441A3 (fr) * 2005-03-24 2006-12-21 Straumann Holding Ag Nouvelle methode de purification de proteines
WO2015166037A1 (fr) * 2014-04-30 2015-11-05 Novozymes A/S Procédé de réduction du taux d'adn d'un bouillon de fermentation
CN106255698A (zh) * 2014-04-30 2016-12-21 诺维信公司 用于减少发酵液的dna含量的方法
US10259841B2 (en) 2014-04-30 2019-04-16 Novozymes A/S Method for reducing the DNA content of a fermentation broth
CN106255698B (zh) * 2014-04-30 2021-08-03 诺维信公司 用于减少发酵液的dna含量的方法
CN105111306A (zh) * 2015-08-28 2015-12-02 北京工业大学 美洲短吻鳄白蛋白的分离方法
WO2019223752A1 (fr) * 2018-05-24 2019-11-28 江苏恒瑞医药股份有限公司 Procédé de préparation d'un précurseur d'insuline humaine recombinée ou d'un analogue associé
WO2022130324A1 (fr) * 2020-12-18 2022-06-23 Glycom A/S Élimination de biomasse par centrifugation

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EP1567657A1 (fr) 2005-08-31
AU2003281982A1 (en) 2004-06-18

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