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WO1990000177A1 - Procede ameliore d'epuration d'insuline - Google Patents

Procede ameliore d'epuration d'insuline Download PDF

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Publication number
WO1990000177A1
WO1990000177A1 PCT/BR1989/000004 BR8900004W WO9000177A1 WO 1990000177 A1 WO1990000177 A1 WO 1990000177A1 BR 8900004 W BR8900004 W BR 8900004W WO 9000177 A1 WO9000177 A1 WO 9000177A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
ion exchange
productivity
flow rate
process according
Prior art date
Application number
PCT/BR1989/000004
Other languages
English (en)
Inventor
Marcos Luiz Dos Mares Guia
Luciano Vilela
Mariza Suzana Santos Pereira Xavier
Heloisa Ribeiro Tunes De Souza
Marcelo Zuculin Junior
Simone Baeta Lage De Oliveira
Original Assignee
Biobras Bioquimica Do Brasil S.A.
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 Biobras Bioquimica Do Brasil S.A. filed Critical Biobras Bioquimica Do Brasil S.A.
Publication of WO1990000177A1 publication Critical patent/WO1990000177A1/fr

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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
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography

Definitions

  • the present invention relates to an improved method for the 5. large scale production of highly purified porcine and Povine insulin crystals.
  • Insulin was discovered in 1921 by two Canadian scientists , Frederick Banting and Charles Best. It is an essential drug 10. in the treatment of the IDDM and is an important adjuvant in the treatment of the NIDDM.
  • Joslin et al (1922) and Karr et al (1931) made the first observations of allergy related to insulin
  • purified insulins must have contamination levels of proinsulin lower than 10 ppm and lower than 1 ppm for pancrea tic polypeptide, measured by RIA. A content of approximately 99% for insulin and desamidoinsulin measured by HPLC, as also 35, discussed.
  • the US 3.069,323 Patent presents a method for the production purified insulin crystals through ion exchange in amino cellu se based anionic resins.
  • the British Patent number 1,140,353 describes an insulin puri cation process through ion exchange by using a carboxymethyl llulose type gel in a high alcohol concentration buffer.
  • the British Patent number 1,285,024 describes an insulin purification process through ion exchange chromatography by using water-miscible organic solvents containing buffer and gels of the Biogel DM, DEAE-Cellulose, Dowex and QAE-Sephadex 5. types.
  • the European Patent Application number 0013826 describes an insulin purification process through ion exchange chromatogra phy by using a high concentration of urea-aqueous buffer in gels of the DEAE-Sephadex types.
  • the process of the present invention is more adequate to industrial production because, by using highe flow rates, cleaning up and regeneration "in situ", a better chromatographic cycle is achieved, with a substantial 35. increase in the daily production of insulin per column volume.
  • the dilution buffer 10. the dilution buffer.
  • the insulin is then eluted with diluti buffer, at a temperature of 0 to 30 degrees with a flow rate up to 2.0 cm/min. After elution of the insulin, the gel washed and equilibrated in the column itself, with buffer c taining a higher concentration of sodium chloride and the di
  • the elution of insulin is controlled through absorvance at 276 nm of collected fractions.
  • the pools corresponding to purified product are precipitated the presence of zinc ions, for later processing through gel f tration or crystallization.
  • the purified product presen a content of insulin higher than 99%. It is important to point out that purity of insulin after ion exchange chromatography, in what respects hormonal contaminant such as Proinsulin, Pancreatic Polypeptide, Somatostatin, Vaso active Intestinal Polypeptide and Glucagon, in each case de 5, pends on the localization of the ion exchange chromatography steps in the whole process of purification, and on the quality and quantity of the starting material.
  • hormonal contaminant such as Proinsulin, Pancreatic Polypeptide, Somatostatin, Vaso active Intestinal Polypeptide and Glucagon
  • Figure 1 shows an elution profile from an ion exchange chroma- 10. tography of insulin on Q-Sepharose Fast Flow in an alcoholic buffer.
  • bovine insulin crystals were dissolved in 350 ml of 0.01M Tris, 7M urea, 0.001M EDTA, pH 8.1 - 8.4 buffer and 15. then applied in a column containing 710 ml of Q-Sepharose Fast Flow (Pharmacia LKB Biotechnology), previously equilibrated with the dilution buffer at a temperature of 4-8 degrees.
  • porcine insulin crystals were dissolved in 350 m of 0.01M TRis, 7M urea, 0.001M EDTA, pH 8.1 - 8.4 buffer an applied in a column containing 630 ml of Q-Sepharose Fast Flo 5. previously equilibrated with the dilution buffer.
  • Elution was carried out in 2100 ml of a sodium chloride grad ent with concentration ranging from 0.07M to 0.20M, in the d lution buffer with a flow rate of 13.0 ml/min at a temperatur of 4-8 degrees. 10. After collection of purified fractions, crystallization, gel filtration in Sephadex G-50-SFSG and one more crystallization 12.8 gms of purified porcine insulin were obtained. The regen ration of the gel was carried out as described in example 1.
  • the precipitate was collected by filtration in a filter press. The recovery of the portion corresponding to the central part of the insulin peak was 71.6%, measured by absorvance at 276 nm. After elution of the insulin peak the column was eluted with 16.5 litres of 0.1M NH C1, 0.8M NaCl, 50% (v/v) Ethanol,

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Le procédé amélioré décrit, qui sert à la production à grande échelle de cristaux d'insuline hautement épurés, utilise, pour la chromatographie par échange ionique de l'insuline, des gels d'une stabilité chimique et physique élevée et permet ainsi d'obtenir un accroissement du débit et de la productivité, définis comme la quantité d'insuline traité par volume de gel par jour.
PCT/BR1989/000004 1988-06-30 1989-03-02 Procede ameliore d'epuration d'insuline WO1990000177A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BRPI8803346 1988-06-30
BR8803346A BR8803346A (pt) 1988-06-30 1988-06-30 Processo aperfeicoado para producao em larga escala de cristais de insulina purificada

Publications (1)

Publication Number Publication Date
WO1990000177A1 true WO1990000177A1 (fr) 1990-01-11

Family

ID=4045112

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/BR1989/000004 WO1990000177A1 (fr) 1988-06-30 1989-03-02 Procede ameliore d'epuration d'insuline

Country Status (2)

Country Link
BR (1) BR8803346A (fr)
WO (1) WO1990000177A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000055184A1 (fr) * 1999-03-15 2000-09-21 Novo Nordisk A/S Chromatographie d'echange d'ions de proteines et de peptides avec un modificateur organique dans l'etape d'elution
KR100341297B1 (ko) * 1997-03-29 2002-11-16 주식회사종근당 재조합활성형프로인슐린의분리.정제방법
US7749955B2 (en) 2003-08-21 2010-07-06 Novo Nordisk A/S Separation of polypeptides comprising a racemized amino acid
US8067554B2 (en) 1999-03-15 2011-11-29 Novo Nordisk A/S Ion exchange chromatography of GLP-1, analogs and derivatives thereof
CN112341535A (zh) * 2019-08-07 2021-02-09 中国科学院大连化学物理研究所 一种采用离子交换色谱法分离纯化制备胰岛素的方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3907676A (en) * 1970-07-28 1975-09-23 Novo Terapeutisk Labor As Process for purifying insulin
EP0085516A2 (fr) * 1982-01-22 1983-08-10 Carlsberg Biotechnology Ltd. A/S Procédé pour remplacement enzymatique d'amino acides en B-30 d'insulines
WO1986005497A1 (fr) * 1985-03-15 1986-09-25 Nordisk Gentofte A/S Derives d'insuline et preparations pharmaceutiques contenant ces derives
GB2173503A (en) * 1985-04-12 1986-10-15 Berlin Chemie Veb Purification of insulin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3907676A (en) * 1970-07-28 1975-09-23 Novo Terapeutisk Labor As Process for purifying insulin
EP0085516A2 (fr) * 1982-01-22 1983-08-10 Carlsberg Biotechnology Ltd. A/S Procédé pour remplacement enzymatique d'amino acides en B-30 d'insulines
WO1986005497A1 (fr) * 1985-03-15 1986-09-25 Nordisk Gentofte A/S Derives d'insuline et preparations pharmaceutiques contenant ces derives
GB2173503A (en) * 1985-04-12 1986-10-15 Berlin Chemie Veb Purification of insulin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Pharmacia, September 1986, FPMG 50-01-329, (SE), "FPLC: media and column quide. High performance separation of biomolecules", pages 6,7,17,18 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100341297B1 (ko) * 1997-03-29 2002-11-16 주식회사종근당 재조합활성형프로인슐린의분리.정제방법
WO2000055184A1 (fr) * 1999-03-15 2000-09-21 Novo Nordisk A/S Chromatographie d'echange d'ions de proteines et de peptides avec un modificateur organique dans l'etape d'elution
US6451987B1 (en) 1999-03-15 2002-09-17 Novo Nordisk A/S Ion exchange chromatography of proteins and peptides
US7276590B1 (en) 1999-03-15 2007-10-02 Novo Nordisk A/S Ion exchange chromatography of proteins and peptides
US8067554B2 (en) 1999-03-15 2011-11-29 Novo Nordisk A/S Ion exchange chromatography of GLP-1, analogs and derivatives thereof
EP2457924A1 (fr) * 1999-03-15 2012-05-30 Novo Nordisk Health Care AG Chromatographie d'échange d'ions de protéines et de peptides
US7749955B2 (en) 2003-08-21 2010-07-06 Novo Nordisk A/S Separation of polypeptides comprising a racemized amino acid
CN112341535A (zh) * 2019-08-07 2021-02-09 中国科学院大连化学物理研究所 一种采用离子交换色谱法分离纯化制备胰岛素的方法

Also Published As

Publication number Publication date
BR8803346A (pt) 1990-02-13

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