EP0000439B1 - Verfahren zur Reinigung eines Insulin enthaltenden, wässerig-äthanolischen Rohextraktes aus Pankreasdrüsen - Google Patents

Verfahren zur Reinigung eines Insulin enthaltenden, wässerig-äthanolischen Rohextraktes aus Pankreasdrüsen Download PDF

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Publication number
EP0000439B1
EP0000439B1 EP78300127A EP78300127A EP0000439B1 EP 0000439 B1 EP0000439 B1 EP 0000439B1 EP 78300127 A EP78300127 A EP 78300127A EP 78300127 A EP78300127 A EP 78300127A EP 0000439 B1 EP0000439 B1 EP 0000439B1
Authority
EP
European Patent Office
Prior art keywords
insulin
ethanol
extract
gel
aqueous ethanol
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.)
Expired
Application number
EP78300127A
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English (en)
French (fr)
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EP0000439A1 (de
Inventor
Willy Henry Jensen
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.)
Laboratorios Leo SA
Original Assignee
Laboratorios Leo SA
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Filing date
Publication date
Application filed by Laboratorios Leo SA filed Critical Laboratorios Leo SA
Publication of EP0000439A1 publication Critical patent/EP0000439A1/de
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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
    • C07K14/625—Extraction from natural sources

Definitions

  • the invention concerns a method of purifying an insulin-containing aqueous ethanolic raw extract from pancreas glands, which method is useful as a purification step in the working up of the extract to pure monocomponent insulin.
  • raw insulin extract is used herein in the usual meaning, i.e. the extract obtained by treating pancreas glands with an aqueous organic solvent, especially aqueous ethanol, usually in a concentration of 60 to 80%, and said term comprises also extracts which, besides being freed from fat, have been subjected to various treatments, for example precipitation of proteins different from insulin by a change of pH value, the so-called pH-8 precipitation, and possibly reverse osmosis, in such a manner, however, that the insulin during these treatments has remained in liquid phase.
  • the term does not comprise insulin-containing solutions formed by dissolution in a solvent of purified solid insulin isolated from the original extract.
  • insulin anti-bodies were produced by the insulin as such.
  • impurities may be accompanying proteins from pancreas distinct from insulin, proinsulin which is a precursor of insulin, intermediate insulin, the dimer, arginine insulin, ethylester insulin, desamido insulin desamidised to various extents, other insulin modifications and coloured substances.
  • the highly purified insulins resulting from the abovementioned known purification methods show a strong decrease in antigenicity but not a complete removal thereof. This is no doubt due to the fact that in spite of the purification steps, the purified preparations still contain substances different from insulin.
  • the said method is based on the recognition that some of the impurities present in insulin are, when using the conventional methods, formed during the recovery itself of insulin, for which reason it is essential to carry out the preparation of insulin in such a manner that the insulin, form the extraction from the pancreas glands until obtention of the final product, is only subjected to conditions of such a nature that they do not cause the formation of decomposition products, aggregates etc.
  • the method is characterized in that the insulin-containing extract prepared from the pancreas glands is worked up to pure insulin in such a way that the insulin is maintained in a solvent environment during the whole processing, until the final recovery of the insulin, the undesired substances being from the beginning of the process until the end removed from the different solvents used.
  • the process can for instance, be carried out by subjecting the extract to a treatment for removal of fat consisting in cooling of the extract to a low temperature, for example between -25°C and -45°C, followed by separation of the crystallized fat; concentrating the extract by means of reverse osmosis, the insulin being simultaneously separated from impurities present in the extract, substances having a larger molecule than insulin as well as substances having a smaller molecule than insulin; washing the insulin-containing concentrate from the reverse osmosis in a reverse osmosis plant for partial removal of coloured substances and salts form the concentrate; subjecting the purified concentrate to further purification by means of ion exchange under conditions at which the insulin remains in liquid phase; and finally recovering the insulin from the concentrated purified solution by precipitation with metal ions, preferably zinc ions, under strong cooling to e.g. -30°C to -45°C.
  • metal ions preferably zinc ions
  • the applicants have therefore set themselves the task of finding a purification method which can supplement or partly replace a treatment of the raw extract with ion exchangers, so that an effective separation is obtained without a loss of insulin or with less loss of insulin than before.
  • the method according to the invention is characterized in that the insulin-containing ethanolic raw extract after having been cooled down to remove fat by crystallization followed by separation of the fat crystals from the extract and optionally having been further pre-purified is subjected at an ethanol concentration being between 60 and 80 per cent by volume, to gel filtration on a gel swollen in ethanol or in aqueous ethanol, preferably with an ethanol concentration of at least 60 per cent by volume, and preferably but not necessarily having the same concentration and the same pH-value as the extractant used, use being made as an eluant likewise of ethanol or of aqueous ethanol, preferably with an ethanol concentration of at least 60 per cent by volume.
  • the gel used is a bead-formed gel possessing both hydrophilic and lipophilic properties. It is a dextran cross-linked with epichlorohydrin and containing hydroxypropyl groups attached to the glucose units of the dextran chains by ether linkages having an exclusion limit of about 10,000 MW and a water regain value of 5.3 to 6.3 ml/g.
  • Such a gel is Sephadex @ LH-60, produced by Pharmacia Fine Chemicals, Uppsala, Sweden.
  • the insulin molecules penetrate the gel particles, being below the exclusion limit of the gel.
  • an insulin fraction is obtained which is totally freed from coloured substances and which contains no or practically no proteins having a larger molecule than insulin or having a smaller molecule than insulin, but only insulin derivatives with practically the same molecular size as the insulin.
  • the yield of insulin is practically 100%.
  • the present method can with special advantage be used in connection with the method described in Danish patent application No. 141/77, the gel filtration supplementing or partly replacing the treatment with ion exchangers described in the specification of said application.
  • the gel filtration according to the present invention is, however, not restricted to the use in connection with said older method but it can be used on any aqueous ethanolic raw extract freed from fat and can constitute either the final purification step before the isolation of solid insulin or an intermediate purification step.
  • the column is prepared in the usual way, the extract - with an ethanol concentration between 60 and 80 per cent by volume - is supplied and the column is filled with the eluant, viz. ethanol or aqueous ethanol.
  • the eluant may be the same as the swelling agent.
  • the pH-value of the liquid phase in the Sephadex bed is preferably between 3 and 8. (Eluates having a pH-value below about 3 are too acid for immediate application to ion exchangers, and at pH-values about 8 the insulin will be partly destroyed).
  • the insulin is eluted and the fraction collected. It is completely free of coloured substances. An almost 100% yield of insulin is obtained.
  • the eluate is further purified, e.g. by means of ion exchanger, and the insulin is finally isolated, for example by precipitation.
  • the fractions containing the insulin peak were collected. They were totally free of coloured substances and contained no or practically no proteins having a larger molecule than insulin or having a smaller molecule than insulin.

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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)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Claims (4)

1. Verfahren zum Reinigen eines Insulin enthaltenden, wässrigethanolischen Rohextraktes von Bauchspeicheldrüsen, dadurch gekennzeichnet, dass der Extrakt nach Abkühlung zwecks Beseitigung von Fett durch Herauskristallisieren und nachfolgende Trennung der Frettkristalle vom Extrakt, und nachdem er eventuell weiter vorgereinigt worden ist, mit einer Ethanolkonzentration von 60 bis 80 Vol-%, einer Gelfiltration an einem Gel unterworfen wird, welches in Ethanol oder wässrigem Ethanol gequollen ist, welches Gel mit Epichlorhydrin vernetztes Dextran, das an den Glucoseeinheiten des Dextrans mittels Ätherbindungen geknüpfte Hydroxypropylgruppen enthält, ist, und das eine Ausschliessungsgrenze von etwa 10.000 MW und eine "water regain value" von 5,3 bis 6,3 ml/g hat, wobei das benutzte Eluiermittel Ethanol oder wässriges Ethanol ist.
2. Verfahren nach Anspruch 1, worin das Gel in wässrigem Ethanol mit einer Konzentration von mindestens 60 Vol.-% gequollen ist.
3. Verfahren nach Anspruch 1 oder 2 worin das Gel in wässrigem Ethanol mit derselben Konzentration und demselben pH-Wert wie die Extraktionsmittel gequollen ist.
4. Verfahren nach einem der vorhergehenden Ansprüche, worin das benutzte Eluiermittel wässriges Ethanol mit einer Ethanolkonzentration von mindestens 60 Vol.-% ist.
EP78300127A 1977-07-08 1978-07-07 Verfahren zur Reinigung eines Insulin enthaltenden, wässerig-äthanolischen Rohextraktes aus Pankreasdrüsen Expired EP0000439B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK773114A DK311477A (da) 1977-07-08 1977-07-08 Fremgangsmaade ved fremstilling af rent insulin
DK3114/77 1977-07-08

Publications (2)

Publication Number Publication Date
EP0000439A1 EP0000439A1 (de) 1979-01-24
EP0000439B1 true EP0000439B1 (de) 1981-09-02

Family

ID=8119244

Family Applications (1)

Application Number Title Priority Date Filing Date
EP78300127A Expired EP0000439B1 (de) 1977-07-08 1978-07-07 Verfahren zur Reinigung eines Insulin enthaltenden, wässerig-äthanolischen Rohextraktes aus Pankreasdrüsen

Country Status (6)

Country Link
EP (1) EP0000439B1 (de)
AT (1) AT368695B (de)
DE (1) DE2860997D1 (de)
DK (1) DK311477A (de)
IE (1) IE47021B1 (de)
IT (1) IT1096920B (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK366380A (da) * 1980-08-28 1982-03-01 Novo Industri As Anvendelsen af grp og salte deraf
DE3726655A1 (de) * 1987-08-11 1989-02-23 Hoechst Ag Verfahren zur isolierung basischer proteine aus proteingemischen, welche solche basischen proteine enthalten
RU2013114350A (ru) 2010-09-16 2014-10-27 Ф.Хоффманн-Ля Рош Аг Новый способ

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2212695A1 (de) * 1972-03-16 1973-09-20 Hoechst Ag Verfahren zur reinigung von insulin und insulinderivaten
US3876623A (en) * 1973-05-09 1975-04-08 Lilly Co Eli Process for purifying insulin
GB1581824A (en) * 1976-01-16 1980-12-31 Leo Sa Lab Preparation of insulin

Also Published As

Publication number Publication date
ATA494578A (de) 1982-03-15
IE47021B1 (en) 1983-11-30
IE781342L (en) 1979-01-08
IT7825460A0 (it) 1978-07-07
IT1096920B (it) 1985-08-26
EP0000439A1 (de) 1979-01-24
DK311477A (da) 1979-01-09
AT368695B (de) 1982-10-25
DE2860997D1 (en) 1981-11-26

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