WO1997018233A1 - Method for producing a correctly folded, biological active recombinant protein - Google Patents

Method for producing a correctly folded, biological active recombinant protein Download PDF

Info

Publication number
WO1997018233A1
WO1997018233A1 PCT/SE1996/001456 SE9601456W WO9718233A1 WO 1997018233 A1 WO1997018233 A1 WO 1997018233A1 SE 9601456 W SE9601456 W SE 9601456W WO 9718233 A1 WO9718233 A1 WO 9718233A1
Authority
WO
WIPO (PCT)
Prior art keywords
igf
protein
cells
buffer
correctly folded
Prior art date
Application number
PCT/SE1996/001456
Other languages
French (fr)
Inventor
Jan-Gunnar Gustafsson
Johan ÖHMAN
Original Assignee
Pharmacia & Upjohn Ab
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 Pharmacia & Upjohn Ab filed Critical Pharmacia & Upjohn Ab
Priority to DE69631906T priority Critical patent/DE69631906T2/en
Priority to JP51878397A priority patent/JP3930051B2/en
Priority to EP96939406A priority patent/EP0952981B1/en
Priority to AU76596/96A priority patent/AU705192B2/en
Priority to AT96939406T priority patent/ATE261985T1/en
Priority to US09/051,941 priority patent/US6605706B1/en
Priority to CA002236751A priority patent/CA2236751C/en
Publication of WO1997018233A1 publication Critical patent/WO1997018233A1/en
Priority to NO19982155A priority patent/NO320175B1/en
Priority to HK00102454A priority patent/HK1023781A1/en

Links

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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
    • 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
    • C07K14/61—Growth hormone [GH], i.e. somatotropin
    • 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
    • C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2

Definitions

  • the present invention relates to a method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of expression of the protein in prokaryotic cells, harvest of the cells, directly solubilization of the cells in a buffer at pH about 8 to 11 and thereafter dilution with water and a diluent.
  • the protein or polypeptide is preferably GH, IGF-I or IGF-II.
  • a general, major problem when recombinant proteins are overproduced in efficient bacterial expression systems is related to the folding of the protein products into their native conformations.
  • Many high level expression system in Escherichia coli results in the production of aggregates of denatured proteins, so called inclusion bodies, which in some cases may be refolded into the wanted native protein.
  • General methods to facilitate and render the refolding effective has been found.
  • One is the use of a class of heat-shock-proteins (HSP) and the other is folding-enzymes. By using HSP, aggregation is avoided and by using the folding enzymes, the speed of refolding is accelerated.
  • HSP heat-shock-proteins
  • IGF-I Human insulin-like growth factor I
  • GH growth hormone
  • IGF-I insulin-like growth factors I and II, Humbel R. E, Eur. J. Biochem 190, 445-462,1990.
  • IGF-I Human recombinant IGF-I has been produced as a secreted product in both Escherichia coli and Saccharomyces cerevisiae. In isolated material from both species, IGF-I is found mainly as miss-folded forms with intermolecular disulphides. In addition, in vitro refolding of reduced IGF-I in the presence of oxygen, has demonstrated that native, miss-matched and aggregated IGF-I accumulate, even under dilute refolding conditions.
  • the refolding yield of recombinant IGF-I was significantly improved by utilising a fused fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A (Samuelsson, E., et al (1991) Bio /Technology Vol. 9, Page 363).
  • ZZ IgG-binding domains
  • the ZZ fusion partner is used to solubilise misfolded molecules before, during and after reduction and reoxidation.
  • the yield of correctly folded IGF-I is shown to be substantially increased but there is still a significant amount of misfolded IGF.
  • Patents and patent applications have also described the problem of misfolded IGF and suggested different improvements.
  • WO 91 /02807 discloses a method for refolding IGF- I in the presence of a fused short positively charged leader sequence, in which amino acids, such as lysine, arginine and histidine are fused at the N- terminus of IGF-I. Inclusion bodies are isolated and solubilized with urea. In WO 93/11240 (Genentech) a method for refolding of insoluble and improperly folded IGF-I is described involving solubilisation of inclusion bodies and refolding in a single buffer system.
  • US 5 151 501 discloses a process for solubilization and naturation of somatropins (Growth hormones) by dispersing somatropin retractile bodies in a solution containing sulfolane and thereafter dilution.
  • WO 9319084 discloses a method for producing active IGF-I is claimed, comprising the steps of expressing in prokaryotic cell, adding a first reducing agent, adding denaturing agent (e.g. urea), adding oxidizing agent (e.g. oxidized gluthatione or cystein) and adding a second reducing agent (e.g. DTT, cystein etc.). Met-IGF-I is expressed.
  • a first reducing agent e.g. urea
  • oxidizing agent e.g. oxidized gluthatione or cystein
  • DTT cystein etc.
  • WO 9506064 discloses a process for increasing the yield of correct refolding of a polypeptide and in which a copper or manganese salt are present during the refolding step and WO 9506059 (Genentech) discloses a method for isolation of cells by adding a phase-forming species to form mutilple aqueous phase.
  • the invention relates to a method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of a) expression of the protein in prokaryotic cells, b) harvest of the cells c) directly solubilization of the cells in a buffer at pH about 8 to 11, , preferably about 8, with a chaotropic agent and a reducing agent and d) dilution with water and a diluent
  • the protein could e.g. be IGF-I, IGF-II or GH.
  • the method preferably comprises the steps of a) expression of an IGF-I-fusion protein in prokaryotic cell system, preferably E Coli, b) harvest of the cells c) directly solubilization of the cells in a buffer at pH 8 to 11, preferably about 8, with a chaotropic agent and a reducing agent d) dilution with water and a diluent e) addition of a cleaving agent and f) purification to produce the biological active IGF-I.
  • the IGF-I-fusion protein is preferably a hybrid Z-IGF-I.
  • the buffer in step c) could be e.g. Tris (Tris [hydroxymethyl]aminomethane hydrochloride) or glycin.
  • the chaotropic agent in step c) is preferably guanidine or urea and guanidine could be used in a concentration of 3-7 M, preferably 5M.
  • the reducing agent in step c) could be e.g. DTT (DL-Dithiothreito) or cystein and the diluent in step d) could be ethanol.
  • pH is preferably reduced below pH 6 and more preferably to pH 3 or below.
  • IGF-I pH is preferably reduced to pH 3 or below between steps d) and e).
  • a concentration step and a buffer exchange between steps d) and e), in which chromatography and/or ion-exchange preferably is used is also claimed.
  • the cleaving agent in step e) in the preparation of IGF-I could be hydroxylamine, an enzyme or any other cleaving agent.
  • the purification steps for the preparation of the pure protein or polypeptide include e.g. cation exchange, RP-HPLC and /or hydrophobic interaction Chromatography (HIC).
  • IGF-I When IGF-I is produced as Z-IGF-I, IGF-I has a better solubility , which means that we can have a higher concentration of reduced IGF-I in solution, giving a high amount of right folded IGF-I which is of most importance for an industrial method for the production of IGF-I.
  • the recombinant human IGF-I (rhIGF-I) used in the experiments was produced in E Coli according to the method described in EP 230 869, example VIII (but in a fermentor) including growing at 37° C for 20 hours.
  • the cell was harvested after fermentation by centrifugation or cross flow filtration. Thereafter the cells were dissolved in:
  • the solubilization solution was diluted with 33.8 L 22.5% ethanol solution, dilution factor 3.
  • the pH was kept at 8.1, under stirring at 15°C.
  • the refolding was stopped after 20 hours by addition of concentrated hydrochloric acid until the pH of the solution was ⁇ 3.1.
  • Solubilization The cell was harvest after fermentation by centrifugation or cross flow filtration. Thereafter the cells were dissolved in:
  • the pH was kept at 10.0, the solubilization was run for 3 hours under stirring at room temperature.
  • the solubilization solution was diluted with 125 mL ethanol, 400 mL water, 66.8 g guanidine-hydrochloride, dilution factor 4.
  • the pH was kept at 10.0, under stirring at room temperature.
  • the refolding was stopped after 20 hours by addition of concentrated hydrochloric acid until the pH of the solution was ⁇ 3.1.
  • the cell was harvested after fermentation by centrifugation or cross flow filtration.
  • EDTA ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate
  • DDT dithiothreitol
  • the pH was kept at 8.0, the solubilization was run for 3 hours under stirring at 15°C.
  • the solubilization solution was diluted with 547 mL ethanol 1786 mL water, dilution factor 3.
  • the pH was kept at 8.1, under stirring at 15°C.
  • the refolding was stopped after 21 hours by addition of concentrated hydrochloric acid until the pH of the solution was ⁇ 3.1.
  • Solubilization 74% (of ZIGF-I from the fermentor) Total, solubilization and refolding: 41%
  • IGF-I was shown to have biological activity in e.g. chick embryo femore and RRA (Radio Receptor Assay).
  • Example 4 first purification step A solution from the refolding step was diluted with WFI (Water for
  • the column was washed with 1 Column volume (CV) WFI, the column was treated with 3 CV of Equilibration buffer and the sample was hereafter applied on the column.
  • the column was first washed with 3 CV of Wash 1 buffer and thereafter with 4 CV Wash 2 buffer.
  • the product was eluted with 5 CV of elution buffer and the column treated with 2 CV of Regeneration buffer and 2 CV of Sanitation buffer.
  • the pH was adjusted to 9.55 (with NaOH) and the temperature was kept at 40° C.
  • the pH was adjusted to 3.30.
  • the pH was adjusted to 9.5 (with HAc) and the temperature was kept at 40° C.
  • the pH was adjusted to 3.35. According to RP-HPLC the yield over this step was 61 % of correctly folded, non oxidized IGF-I.

Landscapes

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

Abstract

The present invention relates to a method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of expression of the protein in prokaryotic cells, harvest of the cells, direct solubilization of the cells in a buffer at pH about 8 to 11 and thereafter dilution with water and a diluent. The protein or polypeptide is preferably GH, IGF-I or IGF-II.

Description

METHOD FOR PRODUCING A CORRECTLY FOLDED, BIOLOGICAL ACTIVE RECOMBINANT PROTEIN
The present invention relates to a method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of expression of the protein in prokaryotic cells, harvest of the cells, directly solubilization of the cells in a buffer at pH about 8 to 11 and thereafter dilution with water and a diluent. The protein or polypeptide is preferably GH, IGF-I or IGF-II.
INTRODUCTION
A general, major problem when recombinant proteins are overproduced in efficient bacterial expression systems is related to the folding of the protein products into their native conformations. Many high level expression system in Escherichia coli results in the production of aggregates of denatured proteins, so called inclusion bodies, which in some cases may be refolded into the wanted native protein. General methods to facilitate and render the refolding effective has been found. One is the use of a class of heat-shock-proteins (HSP) and the other is folding-enzymes. By using HSP, aggregation is avoided and by using the folding enzymes, the speed of refolding is accelerated.
However, not all protein are susceptible for these methods and other solutions to enhance refolding yields have been suggested. In an article by J D Carlson et al in Biotechnology, Vol 10, January 1992, the use of monoclonal antibodies during protein refolding, to enhance the yield of native protein, especially S-Protein, has been disclosed. Another suggested method for the recovering of the native protein is solubilization of the inclusion body protein with a denaturant, such as guanidine or urea and if needed a reduction of the disulphide bond. By dilution or dialysis and reoxidation, the protein can be refolded to the native protein.
Successful refolding, without formation of new inclusion bodies, is generally difficult at high concentrations of the recombinant protein. The best yield is generally achieved at concentrations around 20-200 μg/ml. Refolding is therefore considered to be a very expensive production form that demands a cost intensive drug.
However, the yield of a refolding procedure is unpredictable since the protein product often aggregates or gets modified. In addition, for IGF-I and II, the soluble refolded fraction will contain misfolded species and the overall yield of correctly folded growth factor is rather low (Samuelsson, E., et al (1991) Bio /Technology Vol. 9, Page 363).
In order to increase the yield of correctly folded IGF -I different methods have been proposed.
Human insulin-like growth factor I (IGF-I) is a single-chain peptide growth factor of 70 amino acids, originally isolated from serum. IGF-I is positively regulated by growth hormone (GH) and shows mitogenic effects on many cell types. Therefore, IGF-I is thought to mediate many of the growth promoting effects of GH. In the regions of homology, IGF-I and insulin are 49% homologous, including the six cysteine residues, furnishing three disulphide bridges . The three dimensional structure of IGF-I has been modelled based on the x-ray structure of insulin, and this model has recently been confirmed in the disulphide bridge regions by distance constraints obtained by 2-D NMR spectroscopy of IGF-I (for a review on IGF, see: Insulin-like growth factors I and II, Humbel R. E, Eur. J. Biochem 190, 445-462,1990).
Human recombinant IGF-I has been produced as a secreted product in both Escherichia coli and Saccharomyces cerevisiae. In isolated material from both species, IGF-I is found mainly as miss-folded forms with intermolecular disulphides. In addition, in vitro refolding of reduced IGF-I in the presence of oxygen, has demonstrated that native, miss-matched and aggregated IGF-I accumulate, even under dilute refolding conditions.
The refolding yield of recombinant IGF-I was significantly improved by utilising a fused fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A (Samuelsson, E., et al (1991) Bio /Technology Vol. 9, Page 363). The ZZ fusion partner is used to solubilise misfolded molecules before, during and after reduction and reoxidation. The yield of correctly folded IGF-I is shown to be substantially increased but there is still a significant amount of misfolded IGF.
Patents and patent applications have also described the problem of misfolded IGF and suggested different improvements.
WO 91 /02807 (Amgen) (=US 5158875) discloses a method for refolding IGF- I in the presence of a fused short positively charged leader sequence, in which amino acids, such as lysine, arginine and histidine are fused at the N- terminus of IGF-I. Inclusion bodies are isolated and solubilized with urea. In WO 93/11240 (Genentech) a method for refolding of insoluble and improperly folded IGF-I is described involving solubilisation of inclusion bodies and refolding in a single buffer system.
US 5 151 501 (American Cyanamid) discloses a process for solubilization and naturation of somatropins (Growth hormones) by dispersing somatropin retractile bodies in a solution containing sulfolane and thereafter dilution.
WO 9319084 ( Synergen) discloses a method for producing active IGF-I is claimed, comprising the steps of expressing in prokaryotic cell, adding a first reducing agent, adding denaturing agent (e.g. urea), adding oxidizing agent (e.g. oxidized gluthatione or cystein) and adding a second reducing agent (e.g. DTT, cystein etc.). Met-IGF-I is expressed.
WO 9506064 discloses a process for increasing the yield of correct refolding of a polypeptide and in which a copper or manganese salt are present during the refolding step and WO 9506059 (Genentech) discloses a method for isolation of cells by adding a phase-forming species to form mutilple aqueous phase.
We have now invented a novel, simplified method for the production of correctly folded biological active recombinant protein or polypeptide.
Especially we refer to recombinant IGF-I after expression of Z- IGF-I.
Reference is here given to EP 230 869, especially the examples.
With Escherichia coli expressing the hybrid protein Z-IGF-I, we have achieved very high expression levels (up to 15 g/1 fermentation). Although the method here is described with IGF-I as the preferred polypeptide, it can also be used for recombinant preparation of other polypeptides, when misfolded species and the overall yield of correctly folded polypeptide is rather low.
With our claimed method we can avoid the steps of mechanical disruption of the cells and the isolation and washing of refractile bodies.
Our process is easier than the earlier described and gives a good yield.
THE INVENTION
The invention relates to a method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of a) expression of the protein in prokaryotic cells, b) harvest of the cells c) directly solubilization of the cells in a buffer at pH about 8 to 11, , preferably about 8, with a chaotropic agent and a reducing agent and d) dilution with water and a diluent The protein could e.g. be IGF-I, IGF-II or GH.
When the protein is IGF-I the method preferably comprises the steps of a) expression of an IGF-I-fusion protein in prokaryotic cell system, preferably E Coli, b) harvest of the cells c) directly solubilization of the cells in a buffer at pH 8 to 11, preferably about 8, with a chaotropic agent and a reducing agent d) dilution with water and a diluent e) addition of a cleaving agent and f) purification to produce the biological active IGF-I. The IGF-I-fusion protein is preferably a hybrid Z-IGF-I. The buffer in step c) could be e.g. Tris (Tris [hydroxymethyl]aminomethane hydrochloride) or glycin.
The chaotropic agent in step c) is preferably guanidine or urea and guanidine could be used in a concentration of 3-7 M, preferably 5M. The reducing agent in step c) could be e.g. DTT (DL-Dithiothreito) or cystein and the diluent in step d) could be ethanol.
After step d), pH is preferably reduced below pH 6 and more preferably to pH 3 or below.
For the preparation of IGF-I pH is preferably reduced to pH 3 or below between steps d) and e).
A concentration step and a buffer exchange between steps d) and e), in which chromatography and/or ion-exchange preferably is used is also claimed.
The cleaving agent in step e) in the preparation of IGF-I could be hydroxylamine, an enzyme or any other cleaving agent. The purification steps for the preparation of the pure protein or polypeptide include e.g. cation exchange, RP-HPLC and /or hydrophobic interaction Chromatography (HIC).
When IGF-I is produced as Z-IGF-I, IGF-I has a better solubility , which means that we can have a higher concentration of reduced IGF-I in solution, giving a high amount of right folded IGF-I which is of most importance for an industrial method for the production of IGF-I.
EXAMPLES
The recombinant human IGF-I (rhIGF-I) used in the experiments was produced in E Coli according to the method described in EP 230 869, example VIII (but in a fermentor) including growing at 37° C for 20 hours.
Example 1
Solubilization
The cell was harvested after fermentation by centrifugation or cross flow filtration. Thereafter the cells were dissolved in:
13 L cell solution, wet weight 498 g/L 5 mol/L guanidine-hydrochloride 97 mmol/L Tris-base 159 mmol/L Tris-HCl
2 mmol/L ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate (EDTA) 4 mmol/L dithiothreitol (DDT) Total volume: 16.9 L
The pH was kept at 8.1, the solubilization was run for 3 hours under stirring at 15° C. Refolding
The solubilization solution was diluted with 33.8 L 22.5% ethanol solution, dilution factor 3.
The pH was kept at 8.1, under stirring at 15°C.
The refolding was stopped after 20 hours by addition of concentrated hydrochloric acid until the pH of the solution was <3.1.
RP-HPLC analysis of the concentration of Z-IGF-I in the refolding solution gave correctly folded 3.249 g ZIGF-I/L.
Example 2
Solubilization The cell was harvest after fermentation by centrifugation or cross flow filtration. Thereafter the cells were dissolved in:
154 mL cell solution, wet weight 450 g/L 4.5 mol/L guanidine-hydrochloride 400 mmol/L glycin 0.2% Tween 20
2 mmol/L e ylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate (EDTA)
3 mmol/L dithiothreitol (DDT) Total volume: 200 mL
The pH was kept at 10.0, the solubilization was run for 3 hours under stirring at room temperature.
Refolding
The solubilization solution was diluted with 125 mL ethanol, 400 mL water, 66.8 g guanidine-hydrochloride, dilution factor 4. The pH was kept at 10.0, under stirring at room temperature.
The refolding was stopped after 20 hours by addition of concentrated hydrochloric acid until the pH of the solution was <3.1.
RP-HPLC analysis of the concentration of Z-IGF-I in the refolding solution gave correctly folded 1.38 g ZIGF-I/L.
Example 3 Solubilization
The cell was harvested after fermentation by centrifugation or cross flow filtration.
Thereafter the cells were dissolved in:
900 mL cell solution, wet weight 720 g/L
6 mol/L guanidine-hydrochloride
97 mmol/L Tris-base
159 mmol/L Tris-HCl
2 mmol/L ethylene-dinitro-tetraacetic acid-disodiumsalt-dihydrate (EDTA) 3 mmol / L dithiothreitol (DDT)
Total volume: 1.17 L
The pH was kept at 8.0, the solubilization was run for 3 hours under stirring at 15°C.
Refolding
The solubilization solution was diluted with 547 mL ethanol 1786 mL water, dilution factor 3.
Total volume: 3.6 L
The pH was kept at 8.1, under stirring at 15°C. The refolding was stopped after 21 hours by addition of concentrated hydrochloric acid until the pH of the solution was <3.1.
RP-HPLC analysis of the concentration gave correctly folded 1.32 g Z-IGF- I/L.
Yield
Solubilization: 74% (of ZIGF-I from the fermentor) Total, solubilization and refolding: 41%
Finally purified IGF-I was shown to have biological activity in e.g. chick embryo femore and RRA (Radio Receptor Assay).
Example 4, first purification step A solution from the refolding step was diluted with WFI (Water for
Injection), dilution factor 3 and clarified on a cross flow membrane before applied on a cation exchanger (Pharmacia Biotech BPG 200/500, SP- Sepharose FF, 9.5 Liters, 0.33 m bed high).
The following buffers were used:
Step Buffer pH
Equilibration 100 mM Citric acid /di-Na hydrogen phosphate 3.0
Wash 1 50 mM Citric acid /di-Na hydrogen phosphate 2.9
Wash 2 100 mM di-Na hydrogen- and Na-dihydrogen phosphate 5.9
Elution 100 mM Na hydrogen phosphate 7.5
Regeneration 500m M NaOH
Sanitation 100 mM NaOH
The column was washed with 1 Column volume (CV) WFI, the column was treated with 3 CV of Equilibration buffer and the sample was hereafter applied on the column. The column was first washed with 3 CV of Wash 1 buffer and thereafter with 4 CV Wash 2 buffer.
The product was eluted with 5 CV of elution buffer and the column treated with 2 CV of Regeneration buffer and 2 CV of Sanitation buffer.
According to RP-HPLC the yield over the purification step was 98 % correctly folded IGF-I.
Example 5, cleavage step After the first purification step on a cation ion exchange column, the cleavage of
Z from IGF-I was performed.
To a vessel was added : 40.0 Liters from the cation ion exchanger pool, 1428 grams Sodium diphosphate and 4.0 Liters Hydrozylamine, 50 % sol.
The pH was adjusted to 9.55 (with NaOH) and the temperature was kept at 40° C.
After 3 hours the reaction was stopped by lowering the temperature to 25° C and adding 40.0 Liters concentrated Acetic acid (HAc) and 160 Liters WFI.
The pH was adjusted to 3.30.
According to RP-HPLC the yield over this step was 84 % of correctly folded, non oxidized IGF-I.
Example 6, cleavage step
After the first purification step on a cation ion exchange column, the cleavage of Z from IGF-I was performed.
To a vessel was added : 18.6 Liters from the cation ion exchange pool, 664 grams Sodium diphosphate and 2.17 Liters Hydrozylamine, 50 % sol.
The pH was adjusted to 9.5 (with HAc) and the temperature was kept at 40° C.
After 3 hours the reaction was stopped by lowering the temperature to 25° C and adding 22.3 Liters concentrated HAc and 93 Liters WFI.
The pH was adjusted to 3.35. According to RP-HPLC the yield over this step was 61 % of correctly folded, non oxidized IGF-I.

Claims

1. A method for producing a correctly folded, biological active recombinant protein or polypeptide, comprising the steps of a) expression of the protein or polypeptide in prokaryotic cells b) harvest of the cells c) directly solubilization of the cells in a buffer at pH about 8 to 11 with a chaotropic agent and a reducing agent and d) dilution with water and a diluent.
2. A method according to claim 1 in which the protein is IGF-I, IGF-II or GH.
3. A method according to claim 1 in which the protein is IGF-I and which comprises the steps of a) expression of an IGF-I-fusion protein in prokaryotic cell system, preferably E Coli b) harvest of the cells c) directly solubilization of the cells in a buffer at pH 8 to 11 with a chaotropic agent and a reducing agent d) dilution with water and a diluent e) addition of a cleaving agent and f) purification to produce the biological active IGF-I.
4. Method according to any of claims 1 to 3 in which the IGF-I fusion protein is hybrid Z-IGF-I.
5. Method according to any of claims 1 to 4 in which the buffer in step c) is Tris or glycin.
6. Method according to any of claims 1 to 5 in which pH in step c) is about 8.
7. Method according to any of claims 1 to 6 in which the chaotropic agent in step c) is guanidine or urea.
8. Method according to claim 7 in which guanidine is used in a concentration of 3-7 M.
9. Method according to any of claims 1 to 8 in which the reducing agent in step c) is DTT or cystein.
10. Method according to any of claims 1 to 9 in which the diluent in step d) is ethanol.
11. Method according to any of claims 1 to 10 in which pH is reduced after step d), preferably below pH 6 more preferably to pH 3 or below.
21. Method according to claim 11 in which pH is reduced to pH 3 or below between steps d) and e).
13. Method according to claim 3 in which there is a concentration step and a buffer exchange between steps d) and e).
14. Method according to claim 13 in which chromatography and /or ion- exchange is used.
15. Method according to any of claims 1 to 14 in which cleaving agent in step e) is hydroxylamine.
16. Method according to claim 14 in which purification step f) include cation exchange, RP-HPLC and /or hydrophobic interaction Chromatography
(H1C).
PCT/SE1996/001456 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein WO1997018233A1 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
DE69631906T DE69631906T2 (en) 1995-11-13 1996-11-12 METHOD FOR PRODUCING CORRECTLY FOLDED, BIOLOGICALLY ACTIVE, RECOMBINANT PROTEIN
JP51878397A JP3930051B2 (en) 1995-11-13 1996-11-12 Method for producing correctly folded biologically active recombinant protein
EP96939406A EP0952981B1 (en) 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein
AU76596/96A AU705192B2 (en) 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein
AT96939406T ATE261985T1 (en) 1995-11-13 1996-11-12 METHOD FOR PRODUCING PROPERLY FOLDED, BIOLOGICALLY ACTIVE, RECOMBINANT PROTEIN
US09/051,941 US6605706B1 (en) 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein
CA002236751A CA2236751C (en) 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein
NO19982155A NO320175B1 (en) 1995-11-13 1998-05-12 Process for producing properly folded, biologically active recombinant protein or polypeptide.
HK00102454A HK1023781A1 (en) 1995-11-13 2000-04-26 Method for producing a correctly folded, biological active recombinant protein.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE9504019-2 1995-11-13
SE9504019A SE9504019D0 (en) 1995-11-13 1995-11-13 Method for production of proteins

Publications (1)

Publication Number Publication Date
WO1997018233A1 true WO1997018233A1 (en) 1997-05-22

Family

ID=20400188

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1996/001456 WO1997018233A1 (en) 1995-11-13 1996-11-12 Method for producing a correctly folded, biological active recombinant protein

Country Status (11)

Country Link
US (1) US6605706B1 (en)
EP (1) EP0952981B1 (en)
JP (1) JP3930051B2 (en)
AT (1) ATE261985T1 (en)
AU (1) AU705192B2 (en)
CA (1) CA2236751C (en)
DE (1) DE69631906T2 (en)
HK (1) HK1023781A1 (en)
NO (1) NO320175B1 (en)
SE (1) SE9504019D0 (en)
WO (1) WO1997018233A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042486A1 (en) * 1998-02-23 1999-08-26 G.D. Searle & Co. Method of producing mouse and human endostatin
WO1999050302A1 (en) * 1998-03-31 1999-10-07 Tonghua Gantech Biotechnology Ltd. Chimeric protein containing an intramolecular chaperone-like sequence and its application to insulin production
WO2000075346A1 (en) * 1999-06-09 2000-12-14 Medical Research Council Fusion proteins comprising a fragment of a chaperon polypeptide
US9200030B2 (en) 2006-07-14 2015-12-01 Genentech, Inc. Refolding of recombinant proteins
WO2017075535A1 (en) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Methods of treating neurodegenerative conditions
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2820424B1 (en) * 2001-02-05 2004-01-02 Merieux Oravax ALPA PURIFICATION PROCESS
WO2022097216A1 (en) * 2020-11-05 2022-05-12 シミックホールディングス株式会社 Method for producing recombinant insulin-like growth factor i

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006064A1 (en) * 1993-08-20 1995-03-02 Genentech, Inc. Refolding of polypeptides like recombinant insulin-like growth factor igf-i
WO1995006059A1 (en) * 1993-08-20 1995-03-02 Genentech, Inc. Aqueous multiple-phase isolation of polypeptide

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE8505922D0 (en) * 1985-12-13 1985-12-13 Kabigen Ab CONSTRUCTION OF AN IGG BINDING PROTEIN TO FACILITATE DOWNSTREAM PROCESSING USING PROTEIN ENGINEERING
US5231178A (en) 1991-01-16 1993-07-27 The Salk Institute Biotechnology/Industrial Associates, Inc. Method for the purification of intact, correctly-folded insulin-like growth factor-1
US5288931A (en) * 1991-12-06 1994-02-22 Genentech, Inc. Method for refolding insoluble, misfolded insulin-like growth factor-I into an active conformation
EP0600372B1 (en) * 1992-12-02 1997-02-05 Hoechst Aktiengesellschaft Process for the preparation of proinsulin with correctly linked cystin bridges

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995006064A1 (en) * 1993-08-20 1995-03-02 Genentech, Inc. Refolding of polypeptides like recombinant insulin-like growth factor igf-i
WO1995006059A1 (en) * 1993-08-20 1995-03-02 Genentech, Inc. Aqueous multiple-phase isolation of polypeptide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Volume 12, November 1994, ROGER A. HART et al., "Large Scale, in Situ Isolation of Periplasmic IGF-I from E. Coli", page 1113. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042486A1 (en) * 1998-02-23 1999-08-26 G.D. Searle & Co. Method of producing mouse and human endostatin
AU766154B2 (en) * 1998-02-23 2003-10-09 G.D. Searle & Co. Method of producing mouse and human endostatin
US6653098B1 (en) 1998-02-23 2003-11-25 G. D. Searle & Co. Method of producing mouse and human endostatin
AU766154C (en) * 1998-02-23 2004-05-20 G.D. Searle & Co. Method of producing mouse and human endostatin
WO1999050302A1 (en) * 1998-03-31 1999-10-07 Tonghua Gantech Biotechnology Ltd. Chimeric protein containing an intramolecular chaperone-like sequence and its application to insulin production
US6924120B1 (en) 1998-03-31 2005-08-02 Tonghua Gantech Biotechnology, Ltd. Chaperone protein containing an intramolecular chaperone-like sequence and its application to insulin production
WO2000075346A1 (en) * 1999-06-09 2000-12-14 Medical Research Council Fusion proteins comprising a fragment of a chaperon polypeptide
US9200030B2 (en) 2006-07-14 2015-12-01 Genentech, Inc. Refolding of recombinant proteins
US10105416B2 (en) 2014-02-05 2018-10-23 The Regents Of The University Of California Methods of treating mild brain injury
US10617740B2 (en) 2014-02-05 2020-04-14 The Regents Of The University Of California Methods of treating mild brain injury
US11241483B2 (en) 2014-02-05 2022-02-08 The Regents Of The University Of California Methods of treating mild brain injury
WO2017075535A1 (en) 2015-10-28 2017-05-04 Oxeia Biopharmaceuticals, Inc. Methods of treating neurodegenerative conditions

Also Published As

Publication number Publication date
ATE261985T1 (en) 2004-04-15
SE9504019D0 (en) 1995-11-13
US6605706B1 (en) 2003-08-12
DE69631906D1 (en) 2004-04-22
JP3930051B2 (en) 2007-06-13
EP0952981B1 (en) 2004-03-17
AU705192B2 (en) 1999-05-20
EP0952981A1 (en) 1999-11-03
JP2000500023A (en) 2000-01-11
NO320175B1 (en) 2005-11-07
NO982155D0 (en) 1998-05-12
CA2236751C (en) 2007-01-09
CA2236751A1 (en) 1997-05-22
HK1023781A1 (en) 2000-09-22
DE69631906T2 (en) 2005-01-20
AU7659696A (en) 1997-06-05
NO982155L (en) 1998-05-12

Similar Documents

Publication Publication Date Title
EP0305500B1 (en) Process for the purification of recombinant polypeptides
US7105638B1 (en) Product and process for the production, isolation, and purification of recombinant polypeptide
JP4331270B2 (en) Method for producing peptide using auxiliary peptide
EP0215625B1 (en) Protein recovery
US4966963A (en) Production of proteins in active forms
JPH11130798A (en) Improved method for obtaining insulin precursor having correct cystine linkage
JP2008054693A (en) Methods for purifying authentic igf from yeast hosts
US5789547A (en) Method of producing insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) with correct folding and disulfide bonding
KR20140135959A (en) On-column enzymatic cleavage
EP0545999B1 (en) Process for the preparation of biologically active IGF-1 using IGF-1 having an amino-terminal extension
EP0952981B1 (en) Method for producing a correctly folded, biological active recombinant protein
CA1305285C (en) Production of proteins in active forms
EP0409814B1 (en) Variant proteins and polypeptides possessing enhanced affinity for immobilized-metal affinity matrices
US6586575B1 (en) Purification of somatotropin from transformed microorganisms
Choi et al. Effects of L-arginine on refolding of lysine-tagged human insulin-like growth factor 1 expressed in Escherichia coli
AU684117B2 (en) Use of IGF-BP for refolding of IGF
US7193042B1 (en) Methods for purifying authentic IGF from yeast hosts
EP0578472A2 (en) A process for recovering peptides expressed as fusion proteins
JP2003530316A (en) Extractive regeneration of scrambled single-chain polypeptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP NO US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1996939406

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2236751

Country of ref document: CA

Ref country code: CA

Ref document number: 2236751

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1997 518783

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 09051941

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1996939406

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1996939406

Country of ref document: EP