WO1994024157A1 - A method of detecting the presence of and converting of a polypeptide - Google Patents

A method of detecting the presence of and converting of a polypeptide Download PDF

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Publication number
WO1994024157A1
WO1994024157A1 PCT/DK1994/000157 DK9400157W WO9424157A1 WO 1994024157 A1 WO1994024157 A1 WO 1994024157A1 DK 9400157 W DK9400157 W DK 9400157W WO 9424157 A1 WO9424157 A1 WO 9424157A1
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WO
WIPO (PCT)
Prior art keywords
growth hormone
hgh
derivative
rhgh
hydrophobic
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Application number
PCT/DK1994/000157
Other languages
French (fr)
Inventor
Hans Holmegaard SØRENSEN
Thorkild Christensen
Original Assignee
Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to KR1019950704477A priority Critical patent/KR960701891A/en
Priority to JP6522643A priority patent/JPH08508735A/en
Priority to PL94311198A priority patent/PL311198A1/en
Priority to AU65355/94A priority patent/AU6535594A/en
Priority to EP94913048A priority patent/EP0695310A1/en
Publication of WO1994024157A1 publication Critical patent/WO1994024157A1/en
Priority to BG100068A priority patent/BG100068A/en
Priority to FI955000A priority patent/FI955000A/en
Priority to NO954181A priority patent/NO954181D0/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • 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 hormones [GH] (Somatotropin)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/61—Growth hormones [GH] (Somatotropin)

Definitions

  • a method of detecting the presence of and converting of a polypeptide is a method of detecting the presence of and converting of a polypeptide.
  • the present invention relates to a method of detecting the presence of a hydrophobic derivative of a growth hormone and a method for converting the derivative into the native form of the growth hormone.
  • the growth hormones from man and from the common domestic animals are proteins of approximately 191 amino 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
  • 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.
  • Growth hormone extracted from pituitary glands or growth hormone produced by recombinant techniques is always compared with suitable standards in order to ensure the identity with an authentic product.
  • 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 m- NA level.
  • 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.
  • active ingredients for preparing pharmaceutical preparations 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.
  • the hydrophobic derivative of hu ⁇ man growth hormone disclosed herein may easily be detected by chro atographic methods and may easily be converted into the native form of human growth hormone.
  • the invention relates to a method for detection of the presence of a hydrophobic derivative of a growth hormone comprising an extra sulphur atom as compared to the native growth hormone wherein the growth hormone is subjected to a hydrophobic interaction chromatography eluting the column using a gradient of ammonium sulphate and detecting the presence of the hydrophobic derivative.
  • 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 present invention relates to a method for converting a hydrophobic derivative of a growth hormone into the native form of the growth hormone.
  • the hydrophobic derivati ⁇ ve of human growth hormone may be converted into the native form thereof by treating the derivative with a mercapto compound.
  • the treatment is conveniently carried out in a solution comprising the hydrophobic derivative of human growth hormone in a solvent.
  • hydrophobic derivatives may be converted directly into the native form by a gentle treatment using a mercapto 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.
  • hydrophobic derivative of hGH is isolated before carrying out the convertion into native hGH.
  • the mercapto compound may be any mercapto compound not having an adverse effect on the growth hormone under the reaction conditions. Preferred compounds are such compounds which are able to transform the growth hormone derivative directly into the native form without having to reduce the growth hormone totally breaking both sulphur bridges present in native growth hormone.
  • the mercapto compound may e.g. be cysteine, glutathione, 2-mercapto ethanol or dithiothreitol (DTT) .
  • Preferred compounds are selected from the group consisting of cysteine and glutathione. Most preferred is cysteine.
  • the mercapto compound is normally present in the solution in a concentration of from 0.1 and up to 5 mM. Preferably the concentration is in the interval from 0.5 to 3 mM.
  • the growth hormone is treated with cysteine in a concentration of 1 to 2 mM.
  • 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.
  • 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 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 5 to 10. Solutions being buffered to a pH > 6 are preferred.
  • the solvent is preferably selected from the group consisting of Tris, triethylamine, citric acid, phosphate buffer, and histidine, Tris being the preferred buffer.
  • a preferred buffered solution is buffered to pH 7.5 using 20 mM Tris and 10 mM citric acid.
  • 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.
  • 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, and the feed rate of the paper was 0.50 cm/min.
  • rhGH' 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 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 Speetroscopy
  • ESMS Electro-Spray Mass Speetroscopy
  • 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 1 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 electrospray (also referred to as ion- spray) interface (PI, PI) .
  • Sample introduction was done by a syringe infusion pump (Sage Instruments, Cambridge, MA) 5 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 100.02%, 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 15 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 on a Sep-pak (Stationary phase C 18 from Waters) .
  • the rhGH' showed 20 an increase of mass of 31 ⁇ 2 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 nm. 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 isocratic 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. 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 tops at m/z 1320, 1221, and 1094, whereas the fragmentation of the "lower chain” gave rise to tops 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.
  • 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 3.5 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 appeared after 10 to 15 minutes.
  • Lyophilized rhGH from a sample comprising rhGH 1 was treated as follows for converting the hydrophobic derivative of hGH into native hGH:
  • rhGH' may be transformed into native hGH in the presence of 2mM cysteine.
  • the cleavage may be in a medium comprising cysteine enhancing the formation of the native product having the correct disulphide bridges.
  • the conversion and cleavage is suitably carried out in two stages, first at a high pH for converting the hydrophobic derivative whereafter the pH is lowered in order to effect the cleaving of the amino terminal extension.
  • the starting material had a content of hGH' of 8%.
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal

Abstract

A method of detecting and treating a polypeptide. A hydrophobic derivative of a growth hormone may be detected by hydrophobic interaction chromatography and elution using a gradient of ammonium sulphate followed by peptide mapping. The derivative may be treated with a mercapto compound for converting the derivative into the native form of the growth hormone. Preferably, the growth hormone is human growth hormone, and the mercapto compound is cysteine in a concentration of 1-2 mM.

Description

TITLE
A method of detecting the presence of and converting of a polypeptide.
FIELD OF THE INVENTION
The present invention relates to a method of detecting the presence of a hydrophobic derivative of a growth hormone and a method for converting the derivative 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 amino 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 technigues 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.
Growth hormone extracted from pituitary glands or growth hormone produced by recombinant techniques is always compared with suitable standards in order to ensure the identity with an authentic product.
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 m- NA 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. Thus, a hitherto unknown hydrophobic impurity has been detected in connection with the purification of human growth hormone preparations 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 preparations 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.
It is desirable to find a method for easy detection of the presence of the hydrophobic derivative of growth hormone disclosed herein as well as a need for a method for removing the derivatives from a batch of growth hormone.
It is also possible to remove the hydrophobic derivative by physical separation techniques. However, such a procedure alone is less desirable due to loss of active ingredient.
Thus there is also a need for a process which will ensure a quantitative conversion of the hydrophobic derivative of growth hormone directly into the native product.
BRIEF DESCRIPTION OF THE INVENTION
It has now been found that the hydrophobic derivative of hu¬ man growth hormone disclosed herein may easily be detected by chro atographic methods and may easily be converted into the native form of human growth hormone. Thus, in a first aspect, the invention relates to a method for detection of the presence of a hydrophobic derivative of a growth hormone comprising an extra sulphur atom as compared to the native growth hormone wherein the growth hormone is subjected to a hydrophobic interaction chromatography eluting the column using a gradient of ammonium sulphate and detecting the presence of the hydrophobic derivative.
Hydrophobic interaction chromatography 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.
In a further aspect, the present invention relates to a method for converting a hydrophobic derivative of a growth hormone into the native form of the growth hormone.
It has surprisingly been found that the hydrophobic derivati¬ ve of human growth hormone may be converted into the native form thereof by treating the derivative with a mercapto compound. The treatment is conveniently carried out in a solution comprising the hydrophobic derivative of human growth hormone in a solvent.
It has also been found that such hydrophobic derivatives may be converted directly into the native form by a gentle treatment using a mercapto 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.
According to a still further aspect of the invention the hydrophobic derivative of hGH is isolated before carrying out the convertion into native hGH.
It is preferred to treat the whole batch of growth hormone comprising the hydrophobic derivative of hGH directly without isolating the growth hormone derivative.
The mercapto compound may be any mercapto compound not having an adverse effect on the growth hormone under the reaction conditions. Preferred compounds are such compounds which are able to transform the growth hormone derivative directly into the native form without having to reduce the growth hormone totally breaking both sulphur bridges present in native growth hormone. The mercapto compound may e.g. be cysteine, glutathione, 2-mercapto ethanol or dithiothreitol (DTT) .
Preferred compounds are selected from the group consisting of cysteine and glutathione. Most preferred is cysteine.
The mercapto compound is normally present in the solution in a concentration of from 0.1 and up to 5 mM. Preferably the concentration is in the interval from 0.5 to 3 mM. According to a preferred aspect of the invention, the growth hormone is treated with cysteine in a concentration of 1 to 2 mM. 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. 3_6, 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 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 5 to 10. Solutions being buffered to a pH > 6 are preferred. The solvent is preferably selected from the group consisting of Tris, triethylamine, citric acid, phosphate buffer, and histidine, Tris being the preferred buffer.
A preferred buffered solution is buffered to pH 7.5 using 20 mM Tris and 10 mM citric acid. 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 1
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.
5 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
10 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, and the feed rate of the paper was 0.50 cm/min.
15 The fractions of the eluate comprising the hydrophobic derivative were subjected to peptide mapping as described above.
Alternative Mehod of Detection of Hydrophobic Derivative of Human Growth Hormone
20 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)2SOA, 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
25280 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 Speetroscopy
Recombinant human growth hormone was analyzed by Plasma Desorption Mass Speetroscopy (PDMS) and Electro-Spray Mass Speetroscopy (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 rhGH1 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 electrospray (also referred to as ion- spray) interface (PI, PI) . Sample introduction was done by a syringe infusion pump (Sage Instruments, Cambridge, MA) 5 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 100.02%, 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 15 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 on a Sep-pak (Stationary phase C18 from Waters) . The rhGH' showed 20 an increase of mass of 31±2 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
25 rhGH 22126±2 22125.2 rhGH' 22157±2 rhGH' + DTT 22129±2 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 37°C. 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 nm. 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 isocratic 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
Figure imgf000013_0001
Ser-Val-Glu-Ser-C
Figure imgf000013_0002
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 'f agtment + 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 tops at m/z 1320, 1221, and 1094, whereas the fragmentation of the "lower chain" gave rise to tops 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 hGH1
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
Series I Series II Tube Containing Tube Containing
1 Water 4 Water
2 Pure hGH' 5 Pure hGH' hGH 6 hGH
(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 cysteine in distilled water.
The paper was cut into six pieces (rondels of a diameter of 3.5 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 seen for the test tubes having had water added.
On the papers on test tubes 4 and 6 having had added cysteine, 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 appeared after 10 to 15 minutes.
Example 2
Conversion of Hydrophobic Derivative of Human Growth Hormone into Native Human Growth Hormone
Lyophilized rhGH from a sample comprising rhGH1 was treated as follows for converting the hydrophobic derivative of hGH into native hGH:
A: 4IU hGH were dissolved in 2.5 ml of distilled water.
B: 4IU hGH were dissolved in 2.5 ml of distilled water followed by reduction using 100 μl ercaptoethanol for 10 minutes at ambient temperature. Then the resulting mixture was desalted using PD10 (from Pharmacia,
Sephadex G 25) into 20 mM Tris, pH 8.6. The solution was left for 2h at 4°C and analyzed by hydrophobic interaction chromatography as described in Example 1.
C: 4IU hGH were dissolved in 2.5 ml of 20 mM Tris, pH
8.6. The solution was left for 2h at 4'C and analyzed by hydrophobic interaction chromatography as described in Example 1.
D: 12IU hGH were dissolved in 7.5 ml of the refolding buffer as disclosed in WO 92/03477: 20mM Tris, 2mM EDTA, 2mM Cysteine. The solution was left for 2h at 4°C and analyzed by hydrophobic interaction chromatography as described in Example 1. The sample was then desalted into 2mM His, pH 6.5 and analyzed hydrophobic interaction chromatography as described in Example 1.
The results show that redissolution in the folding buffer (sample D) which is weakly reducing but ensures effective disulphide formation causes transformation of the rhGH1 into native rhGH.
Redissolution in water or Tris, pH 8.5 does not cause conversion (Sample A and C) . If the rhGH' is completely reduced using mercaptoethanol giving a form of hGH identical to the form found in E.Coli cells (but without the presence of hydrogen disulphide in the medium) before homogenization, a correct folding may be obtained in Tris pH 8.5 without the addition of cysteine (sample B) .
As shown above, rhGH' may be transformed into native hGH in the presence of 2mM cysteine. When expressing rhGH as a precursor having an amino terminal extension to be cleaved using DAP1, the cleavage may be in a medium comprising cysteine enhancing the formation of the native product having the correct disulphide bridges. In this case the conversion and cleavage is suitably carried out in two stages, first at a high pH for converting the hydrophobic derivative whereafter the pH is lowered in order to effect the cleaving of the amino terminal extension.
To 4.5 ml of the eluate from the first purification step in normal purification of rhGH in 20mM Tris pH 7.5 comprising chloride ions, 10 mM citric acid at 4°C was added 0.5 ml 20mM cysteine solution in distilled water.
Samples were drawn after 1, 2, 4, 8, 16, 32 and 64 minutes, desalted using a NAP-5 column (Pharmacia) according to the manufactorer's instructions eluting with 25mM Tris. After elution, the pH was adjusted to 7.5, and the contents of 7' peptide was carried out by hydrophobic interaction chromatography as described in Example 1. After 1 minute the peak corresponding to the 7' protein had been reduced by -75%, and after 4 minutes, the top had disappeared totally. Thus, the 7' protein may be converted quantitatively into the native protein by treatment with 2mM cysteine for 4 minutes at 4'C.
Then the pH was adjusted to 4-4.5 for cleaving off an extension, if present.
Example 3
Conversion of Hydrophobic Derivative of Human Growth Hormone into Native Human Growth Hormone
Folding experiments were carried out with a concentration of 2 mM Cys at different pH (4.3, 6.0, 7.5). As starting material was used hGH containing hGH'. 1 ml starting material (cone. 0.7 mg/ l) was adjusted to the chosen pH and mixed with 1 ml 4 mM EDTA, 4 mM Cys, 40 mM Tris, 20 mM Citric acid. At different intervals samples were withdrawn and immediately desalted on a NAP-5 column (Pharmacia) against 20 mM Tris, 10 mM Citric acid adjusted to the chosen pH as above. HIC analysis was carried out using the first-mentioned system in Example 1.
The results of the experiments carried out at ambient temperatures were as follows:
The starting material had a content of hGH' of 8%.
At pH 4.3 the contents of hGH' was reduced to 7.7% after 4 minutes and a sample left overnight still had a content of hGH' of 6%.
At pH 6.0 the contents of hGH1 was reduced to 6.0% after 4 minutes and to 1.7% after 64 minutes. After 20 hours no hGH' was detected.
At pH 7.5 the contents of hGH' was reduced very rapidly. After 1 minute to 2.6%, after 2 minutes to 1.6%, and after 4 minutes, no hGH' was detectable.
The results of carrying out the conversion at a temperature of of 4°C and at pH 7.3 were:
After 2 minutes, the contents of hGH' was reduced to 2.5%, after 4 minutes to 1%, and after 8 minutes, no hGH' could be detected.
This shows that the conversion proceeds rapidly and quantitatively at a pH of 7.5 and more slowly and incomplete at lower pH.
The influence of the temperature is of minor importance. 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 Detecting the Presence of and Converting of a Polypeptide
(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: DK 0445/93
(B) FILING DATE: 20-APR-1993
(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 mercapto compound.
2. A method as claimed in claim 1, wherein the mercapto com¬ pound is selected from the group consisting of cysteine and glutathione, 2-mercapto ethanol and dithiothreitol.
3. A method as claimed in claim 2, wherein the mercapto com- 10 pound is cysteine.
4. A method as claimed in any of claims 1-3, wherein the concentration of the mercapto compound is up to 5 mM.
5. A method as claimed in claim 4, wherein the the mercapto compound is cysteine in a concentration of from 1 to 2 mM.
156. A method as claimed in any of claims 1-5, wherein the growth hormone is human growth hormone.
7. A method for detecting the presence of a hydrophobic derivative of a growth hormone comprising an extra sulphur atom as compared to the native growth hormone wherein the 20 growth hormone is subjected to a hydrophobic interaction chromatography and eluting the column with a salt gradient and detecting the presence of the hydrophobic derivative.
PCT/DK1994/000157 1993-04-20 1994-04-19 A method of detecting the presence of and converting of a polypeptide WO1994024157A1 (en)

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WO2000002900A1 (en) * 1998-07-08 2000-01-20 Pharmacia & Upjohn Ab Method for the production of recombinant peptides with a low amount of trisulfides
WO2004031213A1 (en) * 2002-08-28 2004-04-15 Pharmacia Corporation Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
CN1703421B (en) * 2002-09-20 2010-06-23 法玛西亚公司 Process for decreasing aggregate levels of PEG protein
CN1697839B (en) * 2002-08-28 2010-10-27 法玛西亚公司 Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
WO2011041721A1 (en) 2009-10-02 2011-04-07 Biogen Idec Ma Inc. Methods of preventing and removing trisulfide bonds
US8530191B2 (en) 2004-12-29 2013-09-10 Novo Nordisk A/S Method for preventing formation of trisulfide derivatives of polypeptides
US9562252B2 (en) 2011-05-13 2017-02-07 Biogen Ma Inc. Methods of preventing and removing trisulfide bonds

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MXPA05003121A (en) * 2002-09-20 2005-06-22 Pharmacia Corp Process for decreasing aggregate levels of pegylated protein.

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US4985544A (en) * 1987-08-04 1991-01-15 Kyowa Hakko Kogyo Co., Ltd. Process for renaturing fish growth hormone
WO1992004382A1 (en) * 1990-09-05 1992-03-19 Bunge (Australia) Pty. Ltd. Solubilization of proteins in active forms
US5151501A (en) * 1991-12-20 1992-09-29 American Cyanamid Company Method for solubilization and naturation of somatotropins utilizing sulfolane

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US4985544A (en) * 1987-08-04 1991-01-15 Kyowa Hakko Kogyo Co., Ltd. Process for renaturing fish growth hormone
WO1990002758A1 (en) * 1988-09-12 1990-03-22 Pitman-Moore, Inc. Stable bioactive somatotropins
WO1992004382A1 (en) * 1990-09-05 1992-03-19 Bunge (Australia) Pty. Ltd. Solubilization of proteins in active forms
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Cited By (17)

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US7232894B1 (en) 1998-07-08 2007-06-19 Pharmacia Ab Method for the production of recombinant peptides with a low amount of trisulfides
WO2000002900A1 (en) * 1998-07-08 2000-01-20 Pharmacia & Upjohn 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
WO2004031213A1 (en) * 2002-08-28 2004-04-15 Pharmacia Corporation 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 Method for the preparation of growth hormone and antagonist thereof having lower levels of isoform impurities thereof
CN1703421B (en) * 2002-09-20 2010-06-23 法玛西亚公司 Process for decreasing aggregate levels of PEG protein
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
EP2483289B1 (en) 2009-10-02 2019-03-20 Biogen 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

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