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WO1982004069A1 - Procede de preparation de derives d'insuline - Google Patents

Procede de preparation de derives d'insuline Download PDF

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Publication number
WO1982004069A1
WO1982004069A1 PCT/DK1982/000045 DK8200045W WO8204069A1 WO 1982004069 A1 WO1982004069 A1 WO 1982004069A1 DK 8200045 W DK8200045 W DK 8200045W WO 8204069 A1 WO8204069 A1 WO 8204069A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
amino acid
reaction
enzyme
trypsin
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/DK1982/000045
Other languages
English (en)
Inventor
Insulinlab Nordisk
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nordisk Insulinlaboratorium
Original Assignee
Nordisk Insulinlaboratorium
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 Nordisk Insulinlaboratorium filed Critical Nordisk Insulinlaboratorium
Priority to FI830109A priority Critical patent/FI830109A0/fi
Publication of WO1982004069A1 publication Critical patent/WO1982004069A1/fr
Priority to DK0164/83A priority patent/DK16483D0/da
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to a novel process for the preparation of insulin derivatives.
  • OMPI US-PS No. 3,276,961, Bodanszky et al, 1963 discloses a pro ⁇ cess for the preparation of human insulin from other animal insulins by the action of an enzyme, e.g. carboxypeptidase A or trypsin, in the presence of an excess of threonine.
  • carboxypeptidase A or trypsin e.g. carboxypeptidase A or trypsin
  • human insulin cannot be prepared to any appreciable extent. This is probably due to the fact that trypsin and carboxypeptidase A hydrolyze not only the lysyl-alanine peptide bond (B29-B30) but also the other positions in insulin under the working conditions.
  • Trypsin preferably hydrolyzes the arginyl-glycine peptide bond (B22-B23) rather than the lysyl-alanine bond (B29-B30) , whereas carboxypeptidase A cannot split off the alanine at the C-te ⁇ ninal of the B-chain alone without splitting off the asparagine at the C-terminal of the A-chain. It has la- ter been shown that a specific condition, i.e. reaction in an ammonium bicarbonate buffer solution is necessary in or ⁇ der to hinder the asparagine release, cfr. Hoppe-Seyler's 2. Physiol. Chem., 359, 799-802 (1978).
  • these methods prefer to work with the arginyl-glycine peptide bond and therefore comprise con ⁇ densation of a desoctapeptide-(B23-30) porcine insulin with
  • OMPI a synthetic octapeptide corresponding to the positions B-23-30 in human insulin.
  • first process an alkaline hydrolysis is carried out which is accompanied by unfavourable side reactions.
  • second process comprises a non-specific reaction giving rise to many side reactions an demanding complicated purification procedures. Consequently these processes are not suitable for use on an industrial scale.
  • the coupling is carried out by adding trypsin to a solution of DAI and thre- onine butyl ester (Thr-OBu ) in an organic solvent compris ⁇ ing the organic solvent in an amount of about 60%, whereaf ⁇ ter the reaction proceeds at 37° C. for 20 hours to form (B30-Thr-OBu ) porcine insulin,which is isolated.
  • the protecting group butyl ester is split off with tri- fluoro acetic acid in the presence of anisole.
  • the process of the invention is characterized by treating an insulin with a proteolytic enzyme in the pre ⁇ sence of an excess of an amino acid with protected carboxyl
  • OMPI group and in the presence of an organic solvent to form a B-30-Thr-insulin derivative in an acceptable yield and of a high purity.
  • the reaction may be completed within about 1 hour although longer reaction times may be used.
  • trypsin or an enzyme related thereto can be used as the proteolytic enzyme in the process of the invention trypsin or an enzyme related thereto.
  • the process of the invention is suitable for the preparation of human insulin from porcine insulin.
  • an unpurified in ⁇ sulin product can be used.
  • the unreacted insulin starting product is obtained in a pure form which can be used in insulin preparations for therapeutical use.
  • the unreacted insulin starting product can also be reused as starting material.
  • the human insulin prepared is obtained in a high purity and a reasonable yield in one step from an unpurified porcine insulin product.
  • the process of the invention is suitable for the preparation of insulin on an industrial scale.
  • transformed insulin with protected carboxyl group is formed, and this transformed in ⁇ sulin can be isolated from the reaction mixture and sepa ⁇ rated from unreacted insulin by well known chromatographical methods. By subsequent splitting off of the carboxyl pro ⁇ tecting group the transformed insulin is obtained in a pure form.
  • the carboxyl group of the amino acid introduced in the B-30 position may be protected in the form of different esters and amides, however, when selecting these, regard should be paid to the stability of the insulin at the conditions whic are to be used in the removal of the protecting group.
  • the t-butyl ester is particularly suitable, since the splitting off can be carried out by means of trifluoro acetic acid, but other ester or amide groups can also be removed by care ful, optionally enzymatically catalyzed hydrolysis.
  • the enzyme used in the process of the invention must be ca- pable of splitting lysine carbony1 peptide bonds, and thus use can be made of trypsin or enzymes related thereto, e.g. achromobacterprotease I, the preparation and properties of which are described by Masaki et al., Agric. Biol. Chem. 4__ (1978), pages 1443-1445.
  • the enzyme may have been treated with tosyl-L-phenyl alanine chloromethylketone (TPCK) to eliminate a possible contamination with chymotryp sin-like enzymes.
  • TPCK tosyl-L-phenyl alanine chloromethylketone
  • the enzyme may be used in a dissolved for but may also be bound to an insoluble matrix, e.g. agarose or polyacrylamide or similar polymeric substances.
  • the reaction is carried out under conditions where the en ⁇ zymatically catalyzed hydrolysis is sufficiently suppressed for the peptide forming reaction to proceed.
  • the pH must be between 5 and 10, preferably between 6 and 9.
  • the tempera ⁇ ture should be in the range of 4 to 50 C, preferably 20 to 40° C.
  • the concentration of the reactants, i.e. starting insulin and amino acid ester, should be high, and moreover the ami- no acid ester used should be employed in a large excess, up to a molar ratio of 200:1, preferably in the range of 50:1 to 150:1.”
  • the reaction is carried out in the presence of a water-mis- proficient organic solvent, whereby the hydrolysis reaction is hindered and the solubility of the reactants improved.
  • the organic solvent may be dimethylformamide, acetamide, dimeth-
  • ylsulfoxide, ethanol, glycerin and similar solvents option ⁇ ally in the form of their mixtures.
  • concentration of or ⁇ ganic solvent should be selected in the range of 10% to 80%, preferably 30% to 70%, calculated on the total volume of the reaction mixture.
  • reaction time is chosen dependent on the remaining reac ⁇ tion conditions, but normally it does not exceed 24 hours, usually it is about 1 to about 4 hours.
  • reaction mixture was subjected to gel filtration on a column of Sephadex ⁇ G-50 Superfine (2.6 x 90 cm) in 1 M acetic acid.
  • the fraction containing human insulin ester and unreacted porcine insulin was lyophilised. Yield: 180 mg of product mixture.
  • the product mixture was ion-exchanged at 4 C. on a col ⁇ umn of DEAE cellulose (.-Thatmann DE-52) (5 x 2) equilibrated with 75 ml per hour of a buffer consisting of 0.02 M tris and 7 M urea, adjusted to a pH of 8.1 with hydrochloric acid.
  • OMPI column was eluted for 2.5 hours using the above-mentioned buffer solution, thereafter for 2 hours using the above-men tioned buffer in admixture with 0.0045 moles of sodium chlo ide per liter and finally for 12 hours using the former buf fer in admixture with 0.011 moles of sodium chloride per liter.
  • the eluate contained two proteinaceous main fractions.
  • the fraction eluted at first was identified by high pressure liquid chromatography as being human insulin ester and the fraction eluted thereafter as being unreacted porcine insu lin.
  • the collected fractions were desalted on a column of Sephadex ⁇ G-25 in 0.1 M acetic acid and lyophilised to yield 28 mg of human insulin t-butyl ester and 120 mg of un- reacted porcine insulin.
  • the resultant insulin t-butyl ester (28 mg) was dissolved in 2 ml of trifluoro acetic acid in admixture with 0.2 ml of anisole, and the mixture was held at ambient temperature for 25 minutes.
  • the trifluoro acetic acid was removed in va- cuo on a rotary evaporator, and the residue was extracted with 3 x 10 ml ether to remove the anisole.
  • Upon drying in vacuo 25 mg of pure human insulin were obtained, identified by amino acid analysis and high pressure liquid chromato ⁇ graphy.
  • Example 2 fractionated using the procedure of Example 1 to yield 10 mg of human insulin t-butyl ester and 140 mg of unreacted porcine insulin.
  • the isolated human insulin t-butyl ester (10 mg) was treated in the same way as stated in Example 1 with trifluoro acetic acid and anisole to yield 8.0 mg of pure human insulin.
  • the reaction mixture was purified in the same way as de ⁇ scribed in Example 1. Thereafter, the eluate from the ion- -exchange containing the resultant human insulin methyl es ⁇ ter, identified by high pressure liquid chromatography, was desalted on a column of Sephadex (R. G-25. The column was eluted using 0.05 M aqueous ammonium bicarbonate solution, the resultant eluate was adjusted to a pH of 9.5 using di ⁇ luted ammonia water, whereafter the mixture was held at 25 C. for 48 hours for the complete hydrolysis of the es ⁇ ter.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Des derives d'insuline sont prepares en une seule etape en echangeant l'acide amine a terminaison C de la chaine B dans une insuline par le traitement avec une enzyme proteolitique, telle la tripsine, en presence d'un exces d'un acide amine, par exemple la L-treonine, avec un groupe carboxyle protege et en presence d'un solvant organique, apres quoi le groupe de protection carboxylique est facultativement separe.
PCT/DK1982/000045 1981-05-20 1982-05-19 Procede de preparation de derives d'insuline Ceased WO1982004069A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
FI830109A FI830109A0 (fi) 1981-05-20 1982-05-19 Foerfarande foer framstaellning av insulinderivat
DK0164/83A DK16483D0 (da) 1981-05-20 1983-01-17 Fremgangsmade til fremstilling af insulinderivater

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK2221/81810520 1981-05-20
DK222181 1981-05-20

Publications (1)

Publication Number Publication Date
WO1982004069A1 true WO1982004069A1 (fr) 1982-11-25

Family

ID=8110884

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1982/000045 Ceased WO1982004069A1 (fr) 1981-05-20 1982-05-19 Procede de preparation de derives d'insuline

Country Status (5)

Country Link
EP (1) EP0079362A1 (fr)
JP (1) JPS58500739A (fr)
AU (1) AU8457482A (fr)
NO (1) NO830178L (fr)
WO (1) WO1982004069A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983002772A1 (fr) * 1982-02-08 1983-08-18 Robin Ewart Offord Procede ameliore de preparation d'insuline humaine a partir d'insuline non-humaine
EP0092829A3 (en) * 1982-04-23 1985-08-21 Wako Pure Chemical Industries, Ltd. Process for semi-synthesis of human insulin and alkaline protease for use therein

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0017485A1 (fr) * 1979-04-06 1980-10-15 De Forenede Bryggerier A/S Procédé de production enzymatique de peptides
WO1982000301A1 (fr) * 1980-07-24 1982-02-04 Breddam K Substitution enzymatique de l'amino acide b-30 dans les insulines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0017485A1 (fr) * 1979-04-06 1980-10-15 De Forenede Bryggerier A/S Procédé de production enzymatique de peptides
WO1982000301A1 (fr) * 1980-07-24 1982-02-04 Breddam K Substitution enzymatique de l'amino acide b-30 dans les insulines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Nature Vol 280, 1979-08-02, K MORIHARA ET AL: "Semi-synthesis of human insulin by trypsincatalysed replacement of Ala-B30 by Thr in procine insulin 412-3" *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1983002772A1 (fr) * 1982-02-08 1983-08-18 Robin Ewart Offord Procede ameliore de preparation d'insuline humaine a partir d'insuline non-humaine
EP0087238A1 (fr) * 1982-02-08 1983-08-31 Biogen N.V. Méthode de préparation d'insuline humaine à partir d'insuline non-humaine-
EP0092829A3 (en) * 1982-04-23 1985-08-21 Wako Pure Chemical Industries, Ltd. Process for semi-synthesis of human insulin and alkaline protease for use therein
EP0367302A3 (fr) * 1982-04-23 1991-06-19 Wako Pure Chemical Industries, Ltd. Procédé pour la semi-synthèse d'insuline humaine protéase I d'Achromobacter réticulée soluble dans l'eau pour sa mise en oeuvre et procédé pour la préparation de celle-ci

Also Published As

Publication number Publication date
NO830178L (no) 1983-01-19
JPS58500739A (ja) 1983-05-12
EP0079362A1 (fr) 1983-05-25
AU8457482A (en) 1982-12-07

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