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 monocom p onent 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 antibodies 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.
• amorphous or crystalline insulin prepared in a conventional manner to an extensive further purification, e.g. by ion exchange treatment, gel filtration using a so-called molecular sieve or partition-chromatography (German: Veranders- chromatographie).
• 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, from the extraction from the pancreas glands until the obtaining 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 u p to pure insulin in such a way that the insulin is maintained in dissolved state in a liquid phase 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 0 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 partly removal of colouring substances and salts from 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 a less loss of insulin than before.
• Sephadex ® LH-60 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. Sephadex ® LH-60 is 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 colouring 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 raw 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 insulin is eluted and the fraction collected. It is completely gree of colouring substances. Practically a 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 freed from colouring 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)

Applications Claiming Priority (2)

Publications (2)

Family

ID=8119244

Family Applications (1)

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Cited By (3)

Citations (3)

• 1977
  • 1977-07-08 DK DK773114A patent/DK311477A/da not_active Application Discontinuation
• 1978
  • 1978-07-04 IE IE1342/78A patent/IE47021B1/en unknown
  • 1978-07-07 IT IT25460/78A patent/IT1096920B/it active
  • 1978-07-07 AT AT0494578A patent/AT368695B/de not_active IP Right Cessation
  • 1978-07-07 EP EP78300127A patent/EP0000439B1/de not_active Expired
  • 1978-07-07 DE DE7878300127T patent/DE2860997D1/de not_active Expired

Patent Citations (3)

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