[go: up one dir, main page]

WO1996002570A1 - A method of converting a hydrophobic derivative of a polypeptide into the native form - Google Patents

A method of converting a hydrophobic derivative of a polypeptide into the native form Download PDF

Info

Publication number
WO1996002570A1
WO1996002570A1 PCT/DK1995/000307 DK9500307W WO9602570A1 WO 1996002570 A1 WO1996002570 A1 WO 1996002570A1 DK 9500307 W DK9500307 W DK 9500307W WO 9602570 A1 WO9602570 A1 WO 9602570A1
Authority
WO
WIPO (PCT)
Prior art keywords
growth hormone
sulphite
derivative
hgh
rhgh
Prior art date
Application number
PCT/DK1995/000307
Other languages
French (fr)
Inventor
Thorkild Christensen
Original Assignee
Novo Nordik 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 Nordik A/S filed Critical Novo Nordik A/S
Priority to AU29220/95A priority Critical patent/AU2922095A/en
Publication of WO1996002570A1 publication Critical patent/WO1996002570A1/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
    • C07K1/1136—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 reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • 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

Definitions

  • a method of converting a hydrophobic derivative of a polypeptide into the native form is provided.
  • the present invention relates to a method of converting a hydrophobic derivative of a polypeptide into the native form especially a method of converting a hydrophobic derivative of a growth hormone into the native form of the growth hormone.
  • the growth hormones from man and from the common domestic animals are proteins of approximately 191 a ino acids, syn ⁇ thesized and secreted from the anterior lope of the pituitary gland.
  • Human growth hormone consists of 191 amino acids having a molecular weight of 22125 D.
  • Four cystein residues are present giving rise to two disulfide bridges. The disulfide bridge formed between Cys(53) and Cys(165) results in a major loop, and the disulfide bridge between Cys(182) and Cys(189) results in a minor loop.
  • Growth hormone is a key hormone involved in the regulation of not only somatic growth, but also in the regulation of meta ⁇ bolism of proteins, carbohydrates and lipids.
  • the organ systems affected by growth hormone include the ske ⁇ leton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys.
  • human growth hormone could only be obtained by extraction from the pituitary glands of human cadavers.
  • the very limited supplies of growth hormone restricted the use thereof to longitudinal growth promotion in childhood and puberty for treatment of dwarfism, even though it has been proposed for inter alia treatment of short stature (due to growth hormone deficiency, normal short stature and Turner syndrom) , growth hormone deficiency in adults, infertility, treatment of burns, wound healing, dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, and pseudoarthrosis.
  • growth hormone has been proposed for increasing the rate of growth of domestic animals, for decreasing the proportion of fat in animals to be slaughtered for human con ⁇ sumption, and for increasing the production of milk in lactating animals.
  • human growth hormone is normally produced by expressing a gene coding for human growth hormon, said gene being inserted into a microorganism. The growth hormone is then isolated from the broth, optionally after lysing the microorganisms.
  • the host most commonly used for expressing hGH is E. coli.
  • hGH extracted from pituitaries have been investigated in order to detect aberrant forms and determine their specific activities.
  • a variant single chain form is also produced, wherein the amino acid residues 32-46 are omitted resulting in the socalled 20k form of hGH.
  • This variant is the result of alternate splicing at the -RNA level.
  • variants related to mass, charge, rearrangements, oxidized forms, and split forms are described to be present in hGH- preparations isolated from pituitary glands.
  • hydrophobic impurity was detected in connection with the purification of human growth hormone formulations using Hydrophobic Interaction Chromatography (HIC) under special conditions.
  • HIC Hydrophobic Interaction Chromatography
  • This derivative is normally not detected by any of the other methods employed for testing a sample of human growth hormone including SDS-PAGE, RP-HPLC, IE-HPLC and GPC or by the HIC method run under other conditions.
  • active ingredients for preparing pharmaceutical formulations it is generally preferred to employ active ingredients in a form as pure as possible and, if possible, it is preferred to employ active ingredients being monocomponent compounds.
  • 365-373 discloses a method of transformation of the hydrohpobic derivative into native biosynthetic human growth hormone by treatment with cysteine.
  • hydrophobic derivative of human growth hormone may easily be converted into the native form of human growth hormone by treating the derivative with a sulphite compound.
  • the present invention relates to a method for converting the hydrophobic derivative of a growth hormone into the native form of the growth hormone by treating the derivative with a sulphite compound.
  • the treatment is conveniently carried out in a solution comprising the growth hormone in a solvent.
  • hydrophobic derivatives may be converted directly into the native form by a gentle treatment using a sulphite compound.
  • conversion or "refolding" may according to the invention be carried out using a conventional buffer for refolding of proteins, but without the preceding reduction or denaturation to break the disulfide bridges normally relied upon when refolding proteins.
  • the sulphite compound to be used in accordance with the present invention mey be an alkali metal sulphite such as sodium sulphite or potassium sulphite, ammonium sulphite, or an alkaline-earth metal sulphite such as magnesium sulphite or calcium sulphite.
  • the sulphite compound is generated in situ in a solution for the treatment of the hydrophobic derivative of growth hormone by adding sulphur dioxide to the solution, preferably by bubbling sulphur dioxide through the solution.
  • the concentration of the sulphite compound in the reaction may according to the invention be up to a saturated solution, preferably from 1 to 30 mM, and most preferred about 20 mM.
  • a hydrophobic derivative of hGH is treated with sodium sulphite in a concentration of about 20 mM at pH 7 and at room tempreature.
  • the hydrophobic derivative of growth hormone may optionally be isolated before carrying out the conversion thereof into the corresponding native growth hormone.
  • the solvents used to prepare the solution of derivative of the growth hormone to be treated may e.g. be an aquous buffer buffered at a pH from 3 to 11. Solutions being buffered to a pH > 6 are preferred, and more preferred are solutions buffered to about pH 7.
  • the solvent is preferably selected from the group consisting of Tris, triethylamine, citric acid, phosphate buffer, and histidine.
  • a preferred solution is has pH 7.0.
  • the method of the invention is carried out at temperatures ensuring a suitable rate of conversion of the hydrophobic derivative and a minimum formation of unwanted side products from degradation of the growth hormone.
  • the reaction is carried out at temperatures from the freezing point to about 50"C, preferably at a temperature from about 5'C to about 30 ⁇ C, more preferred form about 15 ⁇ C to about 25'C, and most preferred at room temperature.
  • Hydrophobic interaction chro atography is inter alia described in LC&GC.INTL Vol. 5, No. 11 (1992) 24-29.
  • the HIC may be carried out using a column of phenyl superose in a FPLC apparatus.
  • a convenient apparatus is the FPLC apparatus Phenyl Superose HR 5/5 offered by Pharmacia.
  • the elution may be carried out using suitable salts such as ammonium sulphates and/or ammonium acetate.
  • fractions of the eluate from the HIC comprising the hydrophobic derivative of growth hormone may then be subjected to peptide mapping as disclosed in Chapter 9 in High Performance Liquid Chromatography in Biotechnology, Edited by William S. Hancock, Published by John Wiley & Sons, Inc, 1990.
  • the hydrophobic derivative of growth hormone may be detected by comparing retention times as the fragment comprising a trisulphide bridge has a longer retention time as compared to the corresponding fragment comprising disulphide bridge.
  • the native growth hormone prepared in accordance with the present invention may be used for preparing pharmaceutical formulations in the form of e.g. a solution or a lyophilized powder in any method known per se, e.g. as disclosed in International Patent Publications Nos. WO 89/09614, WO 93/12811, WO 93/12812, WO 93/19776, or WO 94/03198 or in published European patent applications Nos. EP 303746 or EP 374120.
  • the growth hormone prepared in accordance with the present invention may be used for preparing pharmaceutical formulations formulated for administration in any suitable way, e.g. by parenteral or oral administration or administration to a mucosal membrane, e.g. nasal, buccal, sublingual, or vaginal administration.
  • the pharmaceutical formulation may be presented in the form of a dose comprised in a vial or cartridge or any other suitable container such as a prefilled syringe or a pen device.
  • the growth hormone may be formulated in the form of a lyophilized powder to be reconstituted later using conventio ⁇ nal vehicles such as distilled water or water for injection or in the form of a solution comprising growth hormone and a solvent.
  • the solvent used may be water, alcohols such as ethyl, n- propyl or isopropyl, butyl alcohol or mixtures thereof.
  • Such pharmaceutical formulations may furthermore comprise salts for adjusting the tonicity, buffers for adjusting the pH and optionally an excipient in order to facilitate the processing thereof, e.g. lyophilization and the the rapid and complete dissolution of a lyophilized formulation when reconstituting the formulation before use.
  • the formulations may comprise conventional preservatives such as phenol, benzyl alcohol, m-cresol, methyl paraben, propylparaben, benzalconium chloride, and benzethonium chloride.
  • a buffer may e.g. include phosphate, tris, citrate, succinate, acetate, histidine or asparagine buffers.
  • Suitable pH ranges, adjusted with buffer, for hGH formulations are from about 4 to about 8, more preferably from about 5.5 to about 7, most advantageously about 6.0.
  • An excipient may be selected saccharids such as lactose, tre- halose, and sucrose, sugar alcohols such as sorbitol, xylitol, ribitol, myoinisitol, galactitol, mannitol or the like , polysaccharides such as the polymers commercialized as Dextran* products such as Dextran* 40, Dextran* 70 or Dextran* 75, and Ficoll and polyvalent alcohols such as polyethylene glycol or polyvinyl alcohol or a combination of two or more of these.
  • saccharids such as lactose, tre- halose, and sucrose
  • sugar alcohols such as sorbitol, xylitol, ribitol, myoinisitol, galactitol, mannitol or the like
  • polysaccharides such as the polymers commercialized as Dextran* products such as Dextran* 40, Dextran* 70 or Dextran*
  • Neutral salts such as sodium chloride or potassium chloride are optionally used in place of sugars or sugar alcohols.
  • growth hormone may be growth hormone from any origin such as avian, bovine, equine, human, ovine, porcine, salmon, trout or tuna growth hormone, preferably bovine, human or porcine growth hormone, human growth hormone being most preferred.
  • the growth hormone to be treated in accordance with the present invention may be growth hormone isolated from a natural source, e.g. by extracting pituitary glands in a conventional manner, or a growth hormone produced by recombinant techniques, e.g as described in E.B. Jensen and S. Carlsen in Biotech and Bioeng. 35, 1-11 (1990) .
  • the preferred growth hormone is hGH.
  • the "growth hormone” may also be a truncated form of growth hormone wherein one or more amino acid residues has (have) been deleted; an analogue thereof wherein one or more amino acid residues in the native molecule has (have) been substi- tuted by another amino acid residue, preferably a natural amino acid residue, as long as the substitution does not have any adverse effect such as antigenicity or reduced action; or a derivative thereof, e.g having an N- or C-terminal exten ⁇ sion such as Met-hGH, Met-Lys-hGH, Ala-Glu-hGH, Thr-Glu-Ala- Glu-hGH, Ala-Glu-Ala-Glu-hGH, Met-Glu-Ala-Glu-hGH, Met-Phe- Glu-Glu-hGH, Met-Asp-Ala-Asp-hGH, or Met-Glu-Ala-Asp-hGH.
  • the term "dose" of growth hormone refers to that amount that provides therapeutic effect in an administration regimen.
  • the formulations hereof are prepared containing amounts of hGH at least about 0.1 mg/ml, preferably upwards of about 10 mg/ml, preferably from about 1 mg/ml to about 40 mg/ml, more prefer ⁇ ably from about 1 mg/ml to about 25 mg/ml, e.g. from 1 mg/ml to about 5 mg/ml, calculated on the ready-to-use formulation.
  • the ⁇ se formulations contain from about 0.1 mg/mg to about 10 mg/ml, corresponding to the currently contemplated dosage regimen for the intended treatment.
  • the concentration range is not critical to the invention and may be varied by the physician supervising the administration.
  • Fig.l shows a HIC chromatogram for determination of BhGH' in a sample treated with a lmM solution of sodium sulphite
  • Fig.2 shows a HIC chromatogram for determination of BhGH* in a sample treated with a 20mM solution of sodium sulphite.
  • the mass spectrometry showed an increase of mass of 32 dal- tons of the hydrophobic derivative of hGH as compared to na- tive hGH. This can be assigned to the presence of an extra sulphur atom. From the results of the characterization of the hydrophobic growth hormone derivative it was concluded that the derivati ⁇ ve is a human growth hormone having one disulphide bridge (Cys 53-Cys 165) and one trisulphide bridge (Cys 182 - S - Cys 189) and having an amino acid sequence identical to that of native hGH.
  • the buffer system was:
  • Buffer A 1.2M ammonium sulphate, 20mM Tris pH 7.4
  • Buffer B 20mM Tris pH 7.4
  • the buffer was added at a rate of 0.50 ml/min.
  • fractions of the eluate comprising the hydrophobic derivative were subjected to peptide mapping as mentioned above.
  • hGH samples were analyzed on a TSK Ether 5PW (75 x 4.6 mm ID) column at ambient temperature using eluent C and D and a gradient from 40 to 50% eluent D during 30 minutes.
  • Eluent C 2 M (NH 4 ) 2 S0 4 , 20 mM Na 2 HP0 4 x 2H 2 0, pH 6.0.
  • Eluent D 20 mM Na 2 HP0 4 x 2H 2 0, 0.1% PEG, pH 6.0. Detection was performed at 280 nm.
  • Flow 0.5 ml/min.
  • HPLC equipment Data handling and control: Waters 860 Networking computer system, Pumps: Waters pumps model 510, Sample injectors: Waters Wisp 712, Detector: Waters M481 spectrophotometer.
  • the hydrophobic derivative of recombinant human growth hormone was identified by the appearance of an new peak between peak 8 and peak 9 coupled with the disappearance of peak 7 (the 7 peptide) corresponding to amino acid residues 179-191 in a peptide mapping of recombinant human growth hormone (rhGH) .
  • the numbering of the peaks are as disclosed in Chapter 9 in High Performence Liquid Chromatographi (Supra) . Isolation of hydrophobic Derivative of Human Growth Hormone
  • isolation may be carried out by scaling up the procedure described above, or such isolation may e.g. be carried out using the method as described in Bio/Technology 5 (1987) 161-164.
  • PDMS Plasma Desorption Mass Spectroscopy
  • ESMS Electro-Spray Mass Spectroscopy
  • the analysis focused on the detecting the difference between the intact rhGH and rhGH' and the corresponding 7 and 7* tryptic peptides, respectively.
  • the mass of intact rhGH and rhGH' was analyzed by ESMS performed using a API III LC/MS/MS system (Sciex. Thornhill, Ontario, Canada) .
  • the triple quadropople instrument had a mass-to-charge (m/Z) range of 2400 and was fitted with a pneumatically assisted eleetrospray (also referred to as ion- spray) interface (PI, PI) .
  • Sample introduction was done by a syringe infusion pump (Sage Instruments, Cambridge, MA) thorugh a fused capillary (75 ⁇ m i.d.) with a liquid flow rate set at 0.5-1 ⁇ l/min.
  • the instrument m/z scale was calibrated with single charged ammonium adduct ions of poly(propylene glycols) (PPG's) under unit resolution.
  • PPG's poly(propylene glycols)
  • the accuracy of mass determination was in generally better than 0.02%, but low intensity spectra may result in less precise mass determination.
  • Plasma Desorption Mass Spectometry (PDMS) analysis was performed using a BIO-ION 20K 252-Californian time-of-flight instrument (ABI Nordic A/S, Uppsala, Sweden) . Standard procedures for sample application (including in situ reduction using DTT) and analysis were followed (P3,P4). The accuracy of Mass determination was about 0.1%.
  • both rhGH and rhGH' were desalted.
  • the rhGH' showed an increase of mass of 3112 amu as compared with rhGH.
  • the mass of the rhGH' is identical to the calculated mass for reduced hGH.
  • the mass of the tryptic fragment No. 7 of rhGH and rhGH', the 7 and 7' fragments, respectively, were determined by PDMS.
  • the 7 fragment arises from tryptic peptide mapping of rhGH.
  • hGH in a concentration of 1 mg/ml was dialysed against 50 mM Tris, 2 mM CaCl 2 , 6 H 2 0, pH 7.8 for 24 hours af 4°C. 10 ⁇ l of a trypsin solution prepared by dissolving 1 mg trypsin (Bovine, DPCC treated, T-1005 from Sigma) pr. ml. 1 mM HC1, 2 mM CaCl 2 .6H 2 0 was added pr mg. hGH. The digestion was performed in 6 hours at 37 ⁇ C.
  • the digestion product was analysed (25 ⁇ l) using RP-HPLC: Column: Nucleosil RP C18, 250x4 mm, 120 A, 7u (Macherey-Nagel, Art. 720042. Temperature: 45°C. Detection : 215 n . Flow: 1 ml/min. Eluent E: 0.05% (vol/vol) TFA in water, eluent F: 0.05% (vol/vol) TFA in 70% acetonitrile in water. Gradient: 0 to 70% eluent F during 60 minutes. Then the gradient was changed to 100% F during 5 minutes followed by 10 minutes isoeratie elution at 100% F. The gradient was changed back to 0% F during 1 minute and the column was equilibrated for 15 minutes before next run.
  • the 7 fragment of rhGH produced by tryptic cleavage has a calculated mass of 1401 and the Formula I
  • the 7• fragment was isolated by collecting the fraction corresponding to the new peak by RP-HPLC of the trypsin digest as discribed above.
  • a partial Edman Degradation combined with PDMS analysis as well as ESMS was carried out directly on the 7' fragment.
  • PDMS analysis as well as ESMS was carried out directly on the 7' fragment.
  • MS/MS analysis by ESMS gave a series of ionized sequences related to the N-terminal part of the peptide.
  • the MS/MS was carried out using the molecular ion of the 7' peptide having the mass 717amu and a double charge.
  • the fragmentation of the "upper chain” gave rise to peaks at m/z 1320, 1221, and 1094, whereas the fragmentation of the "lower chain” gave rise to peaks at m/z 1247, 1118, 1061, and 974.
  • the conclusion is that the first four amino acid residues in each "chain” - as far as the cystein residues - show normal masses.
  • Filter paper (Whatman glass microfibre filters) was soaked in a 0.1M solution of lead acetate in distilled water, and air dried. Six test tubes were prepared having the contents as stated in the below Table III.
  • test tubes were divided into two series as stated in the below Table IV.
  • the paper was cut into six pieces (rondels of a diameter of 203.5 cm) and placed at the top of the eight test tubes.
  • the rondels were moistened by adding 3-4 drops of distilled water, and the test tubes were left in a water bath at 40 * C for 24 hours.
  • the paper on test tube No. 5 showed a dense black spot ascribed to the formation of lead sulphide.
  • the black spot 5 appeared after 10 to 15 minutes.
  • Lyophilized rhGH from a sample comprising rhGH' was diluted 10 with destilled water to a concentration of lmg hGH/ml.
  • the contents of BhGH' in this preparation was 18%.
  • a 40 mM solution of sodium sulphite pH 7 was prepared by dissolving 50.4 mg sodium sulphite in 10 ml distilled water.
  • 0.5 ml solution of rhGH comprising 1 mg BhGH/ml was mixed with 0.5 ml 2mM solution of sodium sulphite giving a lmM solution of sulphite and was left for 30 minutes at room temperature.

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)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A method of treating a polypeptide. A hydrophobic derivative of a growth hormone may be treated with a sulfite compound for converting the derivative into the native form of the growth hormone. Preferably, the growth hormone is human growth hormone, and the sulfite is sodium sulphite in a concentration of 20 mM.

Description

TITLE
A method of converting a hydrophobic derivative of a polypeptide into the native form.
FIELD OF THE INVENTION
The present invention relates to a method of converting a hydrophobic derivative of a polypeptide into the native form especially a method of converting a hydrophobic derivative of a growth hormone into the native form of the growth hormone.
BACKGROUND OF THE INVENTION
The growth hormones from man and from the common domestic animals are proteins of approximately 191 a ino acids, syn¬ thesized and secreted from the anterior lope of the pituitary gland. Human growth hormone consists of 191 amino acids having a molecular weight of 22125 D. Four cystein residues are present giving rise to two disulfide bridges. The disulfide bridge formed between Cys(53) and Cys(165) results in a major loop, and the disulfide bridge between Cys(182) and Cys(189) results in a minor loop.
Growth hormone is a key hormone involved in the regulation of not only somatic growth, but also in the regulation of meta¬ bolism of proteins, carbohydrates and lipids.
The organ systems affected by growth hormone include the ske¬ leton, connective tissue, muscles, and viscera such as liver, intestine, and kidneys.
Until the development of the recombinant technology and the cloning of the growth hormone gene now giving rise to produc¬ tion of e.g. human growth hormone (hGH) and Met-hGH in indu¬ strial scale, human growth hormone could only be obtained by extraction from the pituitary glands of human cadavers. The very limited supplies of growth hormone restricted the use thereof to longitudinal growth promotion in childhood and puberty for treatment of dwarfism, even though it has been proposed for inter alia treatment of short stature (due to growth hormone deficiency, normal short stature and Turner syndrom) , growth hormone deficiency in adults, infertility, treatment of burns, wound healing, dystrophy, bone knitting, osteoporosis, diffuse gastric bleeding, and pseudoarthrosis.
Furthermore, growth hormone has been proposed for increasing the rate of growth of domestic animals, for decreasing the proportion of fat in animals to be slaughtered for human con¬ sumption, and for increasing the production of milk in lactating animals.
In recombinant techniques human growth hormone is normally produced by expressing a gene coding for human growth hormon, said gene being inserted into a microorganism. The growth hormone is then isolated from the broth, optionally after lysing the microorganisms. The host most commonly used for expressing hGH is E. coli.
hGH extracted from pituitaries have been investigated in order to detect aberrant forms and determine their specific activities. Besides the growth hormone with a molecular weight as mentioned above a variant single chain form is also produced, wherein the amino acid residues 32-46 are omitted resulting in the socalled 20k form of hGH. This variant is the result of alternate splicing at the -RNA level. Also variants related to mass, charge, rearrangements, oxidized forms, and split forms are described to be present in hGH- preparations isolated from pituitary glands.
The development of new assays has enabled detection of derivatives of growth hormone present in very small amounts in preparations and standards. In Eur. J. Biochem. (1994) , 219. 365-373 is disclosed a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli as well as a method of transformation of this derivative into native biosynthetic human growth hormone.
The hydrophobic impurity was detected in connection with the purification of human growth hormone formulations using Hydrophobic Interaction Chromatography (HIC) under special conditions. This derivative is normally not detected by any of the other methods employed for testing a sample of human growth hormone including SDS-PAGE, RP-HPLC, IE-HPLC and GPC or by the HIC method run under other conditions.
For preparing pharmaceutical formulations it is generally preferred to employ active ingredients in a form as pure as possible and, if possible, it is preferred to employ active ingredients being monocomponent compounds.
Eur. J. Biochem. (1994) , 219. 365-373 discloses a method of transformation of the hydrohpobic derivative into native biosynthetic human growth hormone by treatment with cysteine.
It is desirable to have the option to use various methods for removing the derivatives from a batch of growth hormone and especially methods wherein is used reagents being readily obtainable and being as safe as possible to handle.
It is possible to remove the hydrophobic derivative by physical separation techniques. However, such a procedure alone is less desirable due to loss of active ingredient.
BRIEF DESCRIPTION OF THE INVENTION
It has surprisingly been found that the hydrophobic derivative of human growth hormone may easily be converted into the native form of human growth hormone by treating the derivative with a sulphite compound.
Accordingly, the present invention relates to a method for converting the hydrophobic derivative of a growth hormone into the native form of the growth hormone by treating the derivative with a sulphite compound. The treatment is conveniently carried out in a solution comprising the growth hormone in a solvent.
It has been found that the hydrophobic derivatives may be converted directly into the native form by a gentle treatment using a sulphite compound. Thus, the conversion or "refolding" may according to the invention be carried out using a conventional buffer for refolding of proteins, but without the preceding reduction or denaturation to break the disulfide bridges normally relied upon when refolding proteins.
The sulphite compound to be used in accordance with the present invention mey be an alkali metal sulphite such as sodium sulphite or potassium sulphite, ammonium sulphite, or an alkaline-earth metal sulphite such as magnesium sulphite or calcium sulphite.
In accordance with another aspect of the invention, the sulphite compound is generated in situ in a solution for the treatment of the hydrophobic derivative of growth hormone by adding sulphur dioxide to the solution, preferably by bubbling sulphur dioxide through the solution.
The concentration of the sulphite compound in the reaction may according to the invention be up to a saturated solution, preferably from 1 to 30 mM, and most preferred about 20 mM. In a preferred embodiment of the invention, a hydrophobic derivative of hGH is treated with sodium sulphite in a concentration of about 20 mM at pH 7 and at room tempreature.
The hydrophobic derivative of growth hormone may optionally be isolated before carrying out the conversion thereof into the corresponding native growth hormone.
It is preferred to treat the whole batch of growth hormone found to comprise the hydrophobic derivative of GH directly without isolating the growth hormone derivative.
The solvents used to prepare the solution of derivative of the growth hormone to be treated may e.g. be an aquous buffer buffered at a pH from 3 to 11. Solutions being buffered to a pH > 6 are preferred, and more preferred are solutions buffered to about pH 7. The solvent is preferably selected from the group consisting of Tris, triethylamine, citric acid, phosphate buffer, and histidine.
A preferred solution is has pH 7.0.
The method of the invention is carried out at temperatures ensuring a suitable rate of conversion of the hydrophobic derivative and a minimum formation of unwanted side products from degradation of the growth hormone. Normally the reaction is carried out at temperatures from the freezing point to about 50"C, preferably at a temperature from about 5'C to about 30βC, more preferred form about 15βC to about 25'C, and most preferred at room temperature.
Presence of the hydrophobic derivative of a growth hormone comprising an extra sulphur atom as compared to the native growth hormone wherein the growth hormone may be detected by a hydrophobic interaction chromatography wherein the column is eluted using a gradient of ammonium sulphate and detecting the presence of the hydrophobic derivative. Hydrophobic interaction chro atography is inter alia described in LC&GC.INTL Vol. 5, No. 11 (1992) 24-29.
The HIC may be carried out using a column of phenyl superose in a FPLC apparatus. A convenient apparatus is the FPLC apparatus Phenyl Superose HR 5/5 offered by Pharmacia.
The elution may be carried out using suitable salts such as ammonium sulphates and/or ammonium acetate.
The fractions of the eluate from the HIC comprising the hydrophobic derivative of growth hormone may then be subjected to peptide mapping as disclosed in Chapter 9 in High Performance Liquid Chromatography in Biotechnology, Edited by William S. Hancock, Published by John Wiley & Sons, Inc, 1990.
The hydrophobic derivative of growth hormone may be detected by comparing retention times as the fragment comprising a trisulphide bridge has a longer retention time as compared to the corresponding fragment comprising disulphide bridge.
The native growth hormone prepared in accordance with the present invention may be used for preparing pharmaceutical formulations in the form of e.g. a solution or a lyophilized powder in any method known per se, e.g. as disclosed in International Patent Publications Nos. WO 89/09614, WO 93/12811, WO 93/12812, WO 93/19776, or WO 94/03198 or in published European patent applications Nos. EP 303746 or EP 374120.
The growth hormone prepared in accordance with the present invention may be used for preparing pharmaceutical formulations formulated for administration in any suitable way, e.g. by parenteral or oral administration or administration to a mucosal membrane, e.g. nasal, buccal, sublingual, or vaginal administration. The pharmaceutical formulation may be presented in the form of a dose comprised in a vial or cartridge or any other suitable container such as a prefilled syringe or a pen device.
Thus, the growth hormone may be formulated in the form of a lyophilized powder to be reconstituted later using conventio¬ nal vehicles such as distilled water or water for injection or in the form of a solution comprising growth hormone and a solvent.
The solvent used may be water, alcohols such as ethyl, n- propyl or isopropyl, butyl alcohol or mixtures thereof.
Such pharmaceutical formulations may furthermore comprise salts for adjusting the tonicity, buffers for adjusting the pH and optionally an excipient in order to facilitate the processing thereof, e.g. lyophilization and the the rapid and complete dissolution of a lyophilized formulation when reconstituting the formulation before use. The formulations may comprise conventional preservatives such as phenol, benzyl alcohol, m-cresol, methyl paraben, propylparaben, benzalconium chloride, and benzethonium chloride.
A buffer may e.g. include phosphate, tris, citrate, succinate, acetate, histidine or asparagine buffers.
Suitable pH ranges, adjusted with buffer, for hGH formulations are from about 4 to about 8, more preferably from about 5.5 to about 7, most advantageously about 6.0.
An excipient may be selected saccharids such as lactose, tre- halose, and sucrose, sugar alcohols such as sorbitol, xylitol, ribitol, myoinisitol, galactitol, mannitol or the like , polysaccharides such as the polymers commercialized as Dextran* products such as Dextran* 40, Dextran* 70 or Dextran* 75, and Ficoll and polyvalent alcohols such as polyethylene glycol or polyvinyl alcohol or a combination of two or more of these.
Neutral salts such as sodium chloride or potassium chloride are optionally used in place of sugars or sugar alcohols.
In the present context "growth hormone" may be growth hormone from any origin such as avian, bovine, equine, human, ovine, porcine, salmon, trout or tuna growth hormone, preferably bovine, human or porcine growth hormone, human growth hormone being most preferred. The growth hormone to be treated in accordance with the present invention may be growth hormone isolated from a natural source, e.g. by extracting pituitary glands in a conventional manner, or a growth hormone produced by recombinant techniques, e.g as described in E.B. Jensen and S. Carlsen in Biotech and Bioeng. 35, 1-11 (1990) . The preferred growth hormone is hGH.
The "growth hormone" may also be a truncated form of growth hormone wherein one or more amino acid residues has (have) been deleted; an analogue thereof wherein one or more amino acid residues in the native molecule has (have) been substi- tuted by another amino acid residue, preferably a natural amino acid residue, as long as the substitution does not have any adverse effect such as antigenicity or reduced action; or a derivative thereof, e.g having an N- or C-terminal exten¬ sion such as Met-hGH, Met-Lys-hGH, Ala-Glu-hGH, Thr-Glu-Ala- Glu-hGH, Ala-Glu-Ala-Glu-hGH, Met-Glu-Ala-Glu-hGH, Met-Phe- Glu-Glu-hGH, Met-Asp-Ala-Asp-hGH, or Met-Glu-Ala-Asp-hGH.
The term "dose" of growth hormone refers to that amount that provides therapeutic effect in an administration regimen. The formulations hereof are prepared containing amounts of hGH at least about 0.1 mg/ml, preferably upwards of about 10 mg/ml, preferably from about 1 mg/ml to about 40 mg/ml, more prefer¬ ably from about 1 mg/ml to about 25 mg/ml, e.g. from 1 mg/ml to about 5 mg/ml, calculated on the ready-to-use formulation. For use of these compositions in administration to human be¬ ings suffering from hypopituitary dwarfism, for example, the¬ se formulations contain from about 0.1 mg/mg to about 10 mg/ml, corresponding to the currently contemplated dosage regimen for the intended treatment. The concentration range is not critical to the invention and may be varied by the physician supervising the administration.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is described more in detail with reference to the drawings in which
Fig.l shows a HIC chromatogram for determination of BhGH' in a sample treated with a lmM solution of sodium sulphite, and
Fig.2 shows a HIC chromatogram for determination of BhGH* in a sample treated with a 20mM solution of sodium sulphite.
DETAILED DESCRIPTION OF THE INVENTION
The identity of amino acid sequence of the hydrophobic variant of human growth hormone with that of human growth hormone has been determined by tryptic peptide mapping, amino acid sequence analysis of isolated peptide fragments. Furthermore, mass spectrometry has been carried out.
The mass spectrometry showed an increase of mass of 32 dal- tons of the hydrophobic derivative of hGH as compared to na- tive hGH. This can be assigned to the presence of an extra sulphur atom. From the results of the characterization of the hydrophobic growth hormone derivative it was concluded that the derivati¬ ve is a human growth hormone having one disulphide bridge (Cys 53-Cys 165) and one trisulphide bridge (Cys 182 - S - Cys 189) and having an amino acid sequence identical to that of native hGH.
EXPERIMENTAL PART
Example A
Detection of Hydrophobic Derivative of Human Growth Hormone.
The presence of a hydrophobic derivative of recombinant human growth hormone comprising an extra sulphur atom as compared to the native form thereof was detected in accordance with the invention by subjecting the growth hormone to HIC using a FPLC apparatus (Pharmacia) and column of Phenyl Superose HR 5/5 from Pharmacia.
For elution a gradient of ammonium sulphate is used.
The buffer system was:
Buffer A: 1.2M ammonium sulphate, 20mM Tris pH 7.4 Buffer B: 20mM Tris pH 7.4
The chemicals used were all Merck p.a.
The elution was carried out using the following gradient: Time (min.) Buffer
0.0 Cone %B 0.0
1.0 Cone %B 0.0
10.0 Cone %B 100
16.0 Cone %B 100
17.0 Cone %B 0
22.0 End
The buffer was added at a rate of 0.50 ml/min.
The fractions of the eluate comprising the hydrophobic derivative were subjected to peptide mapping as mentioned above.
Alternative Mehod of Detection of Hydrophobic Derivative of Human Growth Hormone
hGH samples were analyzed on a TSK Ether 5PW (75 x 4.6 mm ID) column at ambient temperature using eluent C and D and a gradient from 40 to 50% eluent D during 30 minutes. Eluent C: 2 M (NH4)2S04, 20 mM Na2HP04 x 2H20, pH 6.0. Eluent D: 20 mM Na2HP04 x 2H20, 0.1% PEG, pH 6.0. Detection was performed at 280 nm. Flow: 0.5 ml/min. HPLC equipment: Data handling and control: Waters 860 Networking computer system, Pumps: Waters pumps model 510, Sample injectors: Waters Wisp 712, Detector: Waters M481 spectrophotometer.
The hydrophobic derivative of recombinant human growth hormone (rhGH') was identified by the appearance of an new peak between peak 8 and peak 9 coupled with the disappearance of peak 7 (the 7 peptide) corresponding to amino acid residues 179-191 in a peptide mapping of recombinant human growth hormone (rhGH) . The numbering of the peaks are as disclosed in Chapter 9 in High Performence Liquid Chromatographi (Supra) . Isolation of hydrophobic Derivative of Human Growth Hormone
If it is desired to isolate the hydrophobic derivative from a sample of hGH, such isolation may be carried out by scaling up the procedure described above, or such isolation may e.g. be carried out using the method as described in Bio/Technology 5 (1987) 161-164.
Characterization of Hydrophobic Derivative of Human Growth Hormone by Mass Spectroscopy
Recombinant human growth hormone was analyzed by Plasma Desorption Mass Spectroscopy (PDMS) and Electro-Spray Mass Spectroscopy (ESMS) , respectively.
The analysis focused on the detecting the difference between the intact rhGH and rhGH' and the corresponding 7 and 7* tryptic peptides, respectively.
Determination of Mass of Intact rhGH and rhGH'
The mass of intact rhGH and rhGH' was analyzed by ESMS performed using a API III LC/MS/MS system (Sciex. Thornhill, Ontario, Canada) . The triple quadropople instrument had a mass-to-charge (m/Z) range of 2400 and was fitted with a pneumatically assisted eleetrospray (also referred to as ion- spray) interface (PI, PI) . Sample introduction was done by a syringe infusion pump (Sage Instruments, Cambridge, MA) thorugh a fused capillary (75 μm i.d.) with a liquid flow rate set at 0.5-1 μl/min. The instrument m/z scale was calibrated with single charged ammonium adduct ions of poly(propylene glycols) (PPG's) under unit resolution. The accuracy of mass determination was in generally better than 0.02%, but low intensity spectra may result in less precise mass determination. Plasma Desorption Mass Spectometry (PDMS) analysis was performed using a BIO-ION 20K 252-Californian time-of-flight instrument (ABI Nordic A/S, Uppsala, Sweden) . Standard procedures for sample application (including in situ reduction using DTT) and analysis were followed (P3,P4). The accuracy of Mass determination was about 0.1%.
Before the analysis, both rhGH and rhGH' were desalted. The rhGH' showed an increase of mass of 3112 amu as compared with rhGH. After reduction using DTT, the mass of the rhGH' is identical to the calculated mass for reduced hGH.
The results are shown in the below Table I.
Table I
ESMS Calculated rhGH 2212612 22125.2 rhGH' 2215712 rhGH' + DTT 2212912
Determination of Mass of the tryptic fragment No. 7 of rhGH and rhGH'
The mass of the tryptic fragment No. 7 of rhGH and rhGH', the 7 and 7' fragments, respectively, were determined by PDMS. The 7 fragment arises from tryptic peptide mapping of rhGH.
hGH in a concentration of 1 mg/ml was dialysed against 50 mM Tris, 2 mM CaCl2, 6 H20, pH 7.8 for 24 hours af 4°C. 10 μl of a trypsin solution prepared by dissolving 1 mg trypsin (Bovine, DPCC treated, T-1005 from Sigma) pr. ml. 1 mM HC1, 2 mM CaCl2.6H20 was added pr mg. hGH. The digestion was performed in 6 hours at 37βC. The digestion product was analysed (25 μl) using RP-HPLC: Column: Nucleosil RP C18, 250x4 mm, 120 A, 7u (Macherey-Nagel, Art. 720042. Temperature: 45°C. Detection : 215 n . Flow: 1 ml/min. Eluent E: 0.05% (vol/vol) TFA in water, eluent F: 0.05% (vol/vol) TFA in 70% acetonitrile in water. Gradient: 0 to 70% eluent F during 60 minutes. Then the gradient was changed to 100% F during 5 minutes followed by 10 minutes isoeratie elution at 100% F. The gradient was changed back to 0% F during 1 minute and the column was equilibrated for 15 minutes before next run.
The 7 fragment of rhGH produced by tryptic cleavage has a calculated mass of 1401 and the Formula I
Ile-Val-Gln-Cys-Arg S
(I)
Ser-Val-Glu-Ser-Cys-Gly-Phe Mr: 1401
A difference in mass of 32 amu between the 7 and 7' peptides is observed. After reduction using DTT, identical masses are found for both the 7 and the 7' peptides corresponding to the the calculated mass for the reduced peptide.
The results are shown in the below Table II.
Table II
PDMS Calculated
7 fragment 1401 1401
7 fragment + DTT 617 + 785 618 + 785
7 ' fragment 1433
7 'fragtment + DTT 617 + 785
The 7• fragment was isolated by collecting the fraction corresponding to the new peak by RP-HPLC of the trypsin digest as discribed above. A partial Edman Degradation combined with PDMS analysis as well as ESMS was carried out directly on the 7' fragment. Through four steps it was possible to trace the manual degradation by analyzing the truncated peptide. In each step, two amino acid residues were cleaved off (one from each N- terminal) . The difference in mass of 32 amu between the 7 and 7' peptides was not changed during these four cleavages.
MS/MS analysis by ESMS gave a series of ionized sequences related to the N-terminal part of the peptide. The MS/MS was carried out using the molecular ion of the 7' peptide having the mass 717amu and a double charge. The fragmentation of the "upper chain" gave rise to peaks at m/z 1320, 1221, and 1094, whereas the fragmentation of the "lower chain" gave rise to peaks at m/z 1247, 1118, 1061, and 974. The conclusion is that the first four amino acid residues in each "chain" - as far as the cystein residues - show normal masses.
Thus, the difference in mass of 32 amu between the rhGH and rhGH' seems to be due to the presence of a trisulphide as opposed to the normal disulphide.
Demonstration of the Presence of Extra Sulphur in hGH'
The presence of a trisulphide bridge was demonstrated using lead acetate as described below.
Treatment of rhGH' with cysteine as described below was demonstrated to transform the rhGH' into native rhGH during which the development of hydrogen sulphide was detected.
Filter paper (Whatman glass microfibre filters) was soaked in a 0.1M solution of lead acetate in distilled water, and air dried. Six test tubes were prepared having the contents as stated in the below Table III.
Tabel III
2 tubes of 10 ml containing: Water
2 tubes of 10 ml containing: Pure hGH'
2 tubes of 10 ml containing: hGH - without peak 7'
The test tubes were divided into two series as stated in the below Table IV.
Tabel IV
10 Series I Series II Tube Containing Tube Containing
1 Water 4 Water
2 Pure hGH' 5 Pure hGH' 3 hGH 6 hGH
15 (without peak 7') (without peak 7')
To all tubes of series I was added 2.5 ml of distilled water.
To all tubes of series II was added 2.5 ml of 2.5 mM eysteine in distilled water.
The paper was cut into six pieces (rondels of a diameter of 203.5 cm) and placed at the top of the eight test tubes. The rondels were moistened by adding 3-4 drops of distilled water, and the test tubes were left in a water bath at 40*C for 24 hours.
After 24 hours the paper rondels were examined. No change was 25 seen for the test tubes having had water added. On the papers on test tubes 4 and 6 having had added eysteine, a very faint brownish colouring was observed.
The paper on test tube No. 5 showed a dense black spot ascribed to the formation of lead sulphide. The black spot 5 appeared after 10 to 15 minutes.
Example
Conversion of Hydrophobic Derivative of Human Growth Hormone into Native Human Growth Hormone
Lyophilized rhGH from a sample comprising rhGH' was diluted 10 with destilled water to a concentration of lmg hGH/ml. The contents of BhGH' in this preparation was 18%.
A 40 mM solution of sodium sulphite pH 7 was prepared by dissolving 50.4 mg sodium sulphite in 10 ml distilled water.
To 1 ml 40 mM solution of sodium sulphite was added 19 ml 15 distilled water to obtain a 2mM solution of sodium sulphite.
0.5 ml solution of rhGH comprising 1 mg BhGH/ml was mixed with 0.5 ml 2mM solution of sodium sulphite giving a lmM solution of sulphite and was left for 30 minutes at room temperature.
200.5 ml solution of rhGH comprising 1 mg BhGH/ml was mixed with 0.5 ml 40mM solution of sodium sulphite giving a 20mM solution of sulphite and was left for 30 minutes at room temperature.
After standing, the samples were analyzed by hydrophobic 25 interaction chromatography (HIC) as described in Example A. The resulting chromatograms of the samples are shown in the Figure as Fig. 1 and Fig 2., respectively. In the sample having a concentration of sodium sulphite of ImM the contents of BhGH' was reduced to about 16%, whereas the contents og BhGH' was nearly quantitatively removed in the sample containing 20 mM sodium sulphite.
19
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Novo Nordisk A/S (B) STREET: Novo Alle
(C) CITY: DK-2880 Bagsvaerd
(E) COUNTRY: Denmark
(G) TELEPHONE: +45 44448888
(H) TELEFAX: +45 44490555 (I) TELEX: 37173
(ii) TITLE OF INVENTION: A Method of Converting a Hyrophobic Derivative of a Polypeptide into the Native Form
(iii) NUMBER OF SEQUENCES: 2 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)
(V) CURRENT APPLICATION DATA: APPLICATION NUMBER: (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: lie Val Gin Cys Arg 1 5
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Ser Val Glu Ser Cys Gly Phe 1 5

Claims

1. A method for converting a hydrophobic derivative of a growth hormone into the native form of the growth hormone, wherein the derivative of growth hormone is treated with a
5 sulfite compound.
2. A method as claimed in claim 1, wherein the sulphite com¬ pound is an alkalimetal sulphite, ammonium sulfite, or an alkaline-earth metal sulfite.
3. A method as claimed in claim 1, wherein the sulphite is 10 formed in situ by adding sulphur dioxide to a solution of the hydrophobic derivative of growth hormone.
4. A method as claimed in any of claims 1-3, wherein the concentration of the sulphite compound compound is up to a saturated solution.
155. A method as claimed in claim 4, wherein the the sulphite compound is sodium sulphite in a concentration of from 10 to 30 mM.
6. A method as claimed in any of claims 1-5, wherein the pH is adjusted to 3-11.
207. A method as claimed in claim 6, wherein the pH is adjusted to about 7.
8. A method as claimed in any of claims 1-7, wherein the treatment is carried out at a temperature from about 0°C to about 50°C.
259. A method as claimed in claim 8, wherein the temperature is about room temperature. 10. A method as claimed in any of claims 1-9, wherein the growth hormone is human growth hormone.
PCT/DK1995/000307 1994-07-15 1995-07-14 A method of converting a hydrophobic derivative of a polypeptide into the native form WO1996002570A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU29220/95A AU2922095A (en) 1994-07-15 1995-07-14 A method of converting a hydrophobic derivative of a polypeptide into the native form

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK0850/94 1994-07-15
DK85094 1994-07-15

Publications (1)

Publication Number Publication Date
WO1996002570A1 true WO1996002570A1 (en) 1996-02-01

Family

ID=8098254

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1995/000307 WO1996002570A1 (en) 1994-07-15 1995-07-14 A method of converting a hydrophobic derivative of a polypeptide into the native form

Country Status (4)

Country Link
AU (1) AU2922095A (en)
IL (1) IL114597A0 (en)
WO (1) WO1996002570A1 (en)
ZA (1) ZA955789B (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000002900A1 (en) * 1998-07-08 2000-01-20 Pharmacia & Upjohn Ab Method for the production of recombinant peptides with a low amount of trisulfides
JP2006516021A (en) * 2002-09-20 2006-06-15 ファルマシア コーポレイション Method for reducing the level of aggregation of PEGylated proteins
WO2006069940A1 (en) * 2004-12-29 2006-07-06 Novo Nordisk Health Care Ag Method for preventing formation of trisulfide derivatives of polypeptides
EP1546185A4 (en) * 2002-08-28 2006-09-06 Pharmacia Corp Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
US7470779B2 (en) 2002-09-20 2008-12-30 Pfizer Inc. Process for decreasing aggregate levels of pegylated protein
CN1697839B (en) * 2002-08-28 2010-10-27 法玛西亚公司 Growth hormone and antagonists thereof with reduced isoform impurity levels
WO2011041721A1 (en) 2009-10-02 2011-04-07 Biogen Idec Ma Inc. Methods of preventing and removing trisulfide bonds
US9562252B2 (en) 2011-05-13 2017-02-07 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds
US11370838B2 (en) 2014-07-24 2022-06-28 Genentech, Inc. Methods of conjugating an agent to a thiol moiety in a protein that contains at least one sulfide bond

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0361830A2 (en) * 1988-09-26 1990-04-04 Eli Lilly And Company Purification and refolding of recombinant proteins
WO1992004382A1 (en) * 1990-09-05 1992-03-19 Bunge (Australia) Pty. Ltd. Solubilization of proteins in active forms

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0361830A2 (en) * 1988-09-26 1990-04-04 Eli Lilly And Company Purification and refolding of recombinant proteins
WO1992004382A1 (en) * 1990-09-05 1992-03-19 Bunge (Australia) Pty. Ltd. Solubilization of proteins in active forms

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000002900A1 (en) * 1998-07-08 2000-01-20 Pharmacia & Upjohn Ab Method for the production of recombinant peptides with a low amount of trisulfides
AU755083B2 (en) * 1998-07-08 2002-12-05 Pharmacia Ab Method for the production of recombinant peptides with a low amount of trisulfides
US7232894B1 (en) 1998-07-08 2007-06-19 Pharmacia Ab Method for the production of recombinant peptides with a low amount of trisulfides
US8148331B2 (en) 2002-08-28 2012-04-03 Pfizer Inc. Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
EP1546185A4 (en) * 2002-08-28 2006-09-06 Pharmacia Corp Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
AU2003260045B2 (en) * 2002-08-28 2010-01-07 Pharmacia Corporation Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
CN1697839B (en) * 2002-08-28 2010-10-27 法玛西亚公司 Growth hormone and antagonists thereof with reduced isoform impurity levels
JP2006516021A (en) * 2002-09-20 2006-06-15 ファルマシア コーポレイション Method for reducing the level of aggregation of PEGylated proteins
US7470779B2 (en) 2002-09-20 2008-12-30 Pfizer Inc. Process for decreasing aggregate levels of pegylated protein
JP4865728B2 (en) * 2004-12-29 2012-02-01 ノボ ノルディスク ヘルス ケア アクチェンゲゼルシャフト Method for preventing the formation of trisulfide derivatives of polypeptides
WO2006069940A1 (en) * 2004-12-29 2006-07-06 Novo Nordisk Health Care Ag Method for preventing formation of trisulfide derivatives of polypeptides
US8530191B2 (en) 2004-12-29 2013-09-10 Novo Nordisk A/S Method for preventing formation of trisulfide derivatives of polypeptides
WO2011041721A1 (en) 2009-10-02 2011-04-07 Biogen Idec Ma Inc. Methods of preventing and removing trisulfide bonds
US9005926B2 (en) 2009-10-02 2015-04-14 Biogen Idec Ma Inc. Methods of preventing and removing trisulfide bonds
US10308706B2 (en) 2009-10-02 2019-06-04 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds
US9562252B2 (en) 2011-05-13 2017-02-07 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds
US9790533B2 (en) 2011-05-13 2017-10-17 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds
EP2707383B1 (en) 2011-05-13 2018-04-18 Biogen MA Inc. Methods of preventing and removing trisulfide bonds
EP3388443A1 (en) 2011-05-13 2018-10-17 Biogen MA Inc. Methods of preventing and removing trisulfide bonds
US10590454B2 (en) 2011-05-13 2020-03-17 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds
US11370838B2 (en) 2014-07-24 2022-06-28 Genentech, Inc. Methods of conjugating an agent to a thiol moiety in a protein that contains at least one sulfide bond

Also Published As

Publication number Publication date
ZA955789B (en) 1996-03-11
IL114597A0 (en) 1995-11-27
AU2922095A (en) 1996-02-16

Similar Documents

Publication Publication Date Title
Chrétien et al. Isolation, purification, and characterization of γ-lipotropic hormone from sheep pituitary glands
EP0383472B1 (en) Insulin analogs
US5700662A (en) Process for preparing insulin analogs
Nilsson et al. Integrated production of human insulin and its C-peptide
US20100210815A1 (en) Insulin production methods and pro-insulin constructs
Maghuin‐Rogister et al. Luteinizing Hormone: The Primary Structures of the β‐Subunit from Bovine and Porcine Species
Sairam et al. Human pituitary lutropin: isolation, properties, and the complete amino acid sequence of the β-subunit
Nyberg et al. Human pituitary prolactin. Isolation and characterization of three isohormones with different bioassay and radioimmunoassay activities
ZA200102780B (en) Method for obtaining active β-NGF.
WO1996002570A1 (en) A method of converting a hydrophobic derivative of a polypeptide into the native form
Soedjanaatmadja et al. Demonstration by mass spectrometry that pseudo-hevein and hevein have ragged C-terminal sequences
Gram et al. A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli
Li et al. β‐endorphin: isolation, amino acid sequence and synthesis of the hormone from horse pituitary glands
AU6535594A (en) A method of detecting the presence of and converting of a polypeptide
Smith et al. The use of high-performance liquid chromatography in the isolation and characterisation of mouse and rat epidermal growth factors and examination of apparent heterogeneity
EP0381433B1 (en) A method for the production of glucagon
WO1991010684A1 (en) Oxidized variants of gm-csf
KR100443890B1 (en) Process for purifying human growth hormone from recombinant e. coli
Iwakura et al. Expression and purification of growth hormone-releasing factor with the aid of dihydrofolate reductase handle
OSHIKA et al. Human parathyroid hormone: efficient synthesis in Escherichia coli using a synthetic gene, purification and characterization
Olson et al. Production of a biologically active variant form of recombinant human secretin
WADE et al. DIABETOGENIC ACTION OF HUMAN GROWTH HORMONE Synthesis and Activity of C‐terminal Fragments
Martinat et al. Purification and partial characterization of growth hormone from the dromedary (Camelus dromedarius)
KR0132003B1 (en) Process for preparation of recombinant proteins by use of streptomyces thermonitrificans aminopeptidase
Bell et al. Isolation, purification, and properties of porcine calcitonins

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AU BB BG BR BY CA CN CZ EE FI GE HU IS JP KE KG KP KR KZ LK LR LT LV MD MG MN MW MX NO NZ PL RO RU SD SG SI SK TJ TM TT UA UG UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

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
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 96504607

Format of ref document f/p: F