• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A process for enzymatic replacement of the B-30 amino acid in insulins, in which the insulin Ins-X, wherein Ins represents the insulin molecule except for X which represents the B-30 amino acid, is reacted with an amine component selected from amino acids, and their amides and esters, of the formulae… H-B-OH… H-B-NR<1>R<2>… H-O-OR<3>… H-B-SR<3>… H-B-SeR<3>… wherein B is an amino acid residue; either R<1> and R<2> are independently selected from hydrogen, amino, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl and aralkyl or NR<1>R<2> is a heterocyclic group which may contain a further hetero atom; and R<3> is alkyl, cycloalkyl, aryl, heteroaryl or aralkyl; in the presence of an L-specific serine or thiol carboxy-peptidase enzyme, in an aqueous solution or dispersion having a pH from about 7 to 10.5, to form an insulin derivative of the formulae… Ins-B-OH, Ins-B-NR<1>R<2>, Ins-B-B-NR<1>R<2>… Ins-B-OR<3>, Ins-B-SR<3> or Ins-B-SeR<3>… wherein Ins, B, R<1>, R<2> and R<3> are as defined above; and, if desired, cleaving any -NR<1>R<2>, -B-NR<1>R<2>, -OR<3>, -SR<3> or -SeR<3> group. The invention allows human insulin to be prepared from porcine insulin and threonine.
    公开号:SU1611217A3
    申请号:SU843825251
    申请日:1984-12-18
    公开日:1990-11-30
    发明作者:Клаус Бреддам；Джек Таанинг Ехансен；Фред Видмер
    申请人:Карсберг Биотехнолоджи Лтд.А/С (Фирма)；
    IPC主号:
    专利说明:

    The invention relates to biotechnology and can be used to produce insulin in about 1 time from pig insulin.
    The purpose of the invention is to improve the method achieved by eliminating labor-intensive pretreatment of insulin and removing protective chemical groups, as well as reducing the time required for the reaction.
    The method is based on the yeast carboxypeptidase-Y property converting pig insulin into human insulin by exchanging alanine residue, which is in position B 30, for trealin (the threonine source is a threonine derivative in the incubation medium) in one stage without isolating the intermediate product.
    The method is carried out by the following
    I
    Insulin derivatives ins-tpe-III ins-tre-tre-ot VD ins-tre-oCH, where ins-de-tre (B 30) is insulin, is incubated with yeast carboxypepidase Y and derivatives of threonine at rL 7.5-10.0. Preferably, the carcass derivative is tre-scammamide. The reaction is carried out in an aqueous medium, which can contain up to 50% of the volume of organic solvent, and the choice of solvent depends on the solubility of the components of the reaction medium. The reaction medium can also contain urea or chloroprat guanidine in concentrations up to 3 M. The optimal concentration of insus. Is 0.002-0.05 M, the threonine enzyme is 0.05-3.0 M, the temperature is iU and c. These parameters may vary within 1 SHOW limits and are determined experimentally.
    YU
    them
    CM
    Example 1, Lrear1 chenne.1 CHUHCV insulin in human HHcyjBiH using interferon teroninamine as an amine comonent and with an isolation of 5 intermediates: insulinamide and cholesterol deamidization according to the invention
    To a solution of Dink-free porcine insulin (2 m10 in 2 mM EDTL III 0.1 M KC1 and 1.5 M hporgi.; Guataine fata containing 0.5 M threonine amide (5 L-Tpe-NH,) iri rP 7.5 and 25 ° C, carboxypeptidase-Y (15 µM) is added. The pH value during the reaction is maintained-- 15 It is the result of the addition of 0.5 M sodium hydroxide using pH-statas. During the reaction, the ajuiKBOTbi reaction mixture is taken in different times, and the reaction itself remains as a result of quenching 2 hours later by adjusting the pH to 1.0-2.0 by adding a 6 M solution of hydrochloric acid, Fracd: sho, containing insulin, section from chromatography on a 1x30 cm column filled with G-50 fine using 1 M solution of acetic acid as a solvent, and lyophilized in an amino acid composition of lyophilized insulin described as described in the preceding paragraph of the amino acid composition of lyophilized insulin that 78% of porcine insulin is consumed during the reaction 35
    The days for further analysis of the reagents contained in the insulin sample are chromatographed on ion exchange Sephadex-DND. A sample of free-flowing insuds 40 (75 mg) was dissolved in 0.01 M TRIS, 2.5 M urea, 0.05 M NaCl, pryH7.5 and chromatographed on a column filled with Sephadex-DKA1 A-25 (2.5x x2 , 5 than) using as the 4 solvent the same buffer solution. Insulin is eluted with sodium chloride solutions with a concentration gradient. traction from 0.05 to 0.30 M in the same buffer, collect fractions of 8 ml on a column filled with Sephadex G-25 and lyophilize them
    The profile consists of three peaks. In addition, the compositions of aot (no-acids and the composition of substances of the peaks as a result of treatment with carboxypeptidase are determined (see
    t-abl „1) about
    Since only the end of the chain B is involved in these reactions, insulin
    as model 1: and porcine insulin isiol1, it is 11 consecutive;: ьн - procis-ala-OI. Similarly, the abbreviations for other H-insulin insulins are -npo-jni3-Tpe-OH for human insulin (-propyl-tre-NIL) for human 1 amide insulin and so on. At RP 7.5, which is used during the reaction in which the amidase activity of carboxesteptidase-U is usually lower, the check of its peptidase activity, formed by peptide 1id, is significantly stable, since peak I corresponds to approximately 20% of the total 1I sulphylum sample,
    During the reaction, approximately 75% of the initial porcine insulin is converted. However, although the threonine content in peak I (3.65) is greater than 3.0 corresponding to human insulin, it is obvious that the initial product of transference (-product-tre-HH) is not sufficiently stable; 1-site oligomerization to form (-product-trash
    At first glance, the formation of a mixture of intermediates may indicate that as a result of the carboxypeptidase-Y carboxypeptidase-Y stage described earlier, no human and}) human sulins are formed, since as a result of the deamidation, it is obvious that a mixture of -pro jn-13-ter OH and -product-tre-tre-OP ,, However, when treating the substance of peak I in order to deamidate according to the invention 10 MKl l carboxypeptidase-Y at pH 10.0 in 0.1 M HCl, 2 mM EDTA for 20 min get almost pure pure human insulin "This experience is about flows as follows
    After separating the reacted insulin from the enzyme and low molecular weight products by chromatography on Sephadex G-50, the resulting chromatographic product on DEAE-Sefoads A-25 using a method similar to that described above, the product of the eluant is determined in peak II, which was expected while the unreacted product (less than 10%) of the product is recovered at peak I. Peak III is completely absent, i.e. insulin derivatives that do not contain lysine do not form in the course. As a result of treatment with carboxypeptidases A and Y and trypsin, this reaction affects TLVILKO on soda.
    51
    threonine to a large extent. This is consistent with the expected lack of peptidase activity of carboxypeptidase-Y at a given value.
    The composition of the amino acids for peak II, obtained by the deamidation reaction, is close to the insulin composition. In humans: 3.87, Ala 1.05, LIZ 0.98. Treatment of the deamidation product with carboxypeptidases Y and A and trypsin indicates that the sample contains 90-96% of pure human insulin on the assumption that the insulin derivative β-pro-lys-tre-tre-III reacts practically only when there is hydrolase activity to the amino acid pentidyl amide, and pro-lpz-tre-SH2 reacts mainly with the presence of activity. porcine insulin to human insulin is approximately 30% based on npeBjia insulin
    T a b. d and c
    ten
    16112
    off
    but
    15
    20
    25
    176
    Example 2 of human insideline aid (ins-tre-III) in an amount of 11 mg is dissolved in 1 mM EDTA and 2 mM CU (2 ml), the pH is adjusted to 9.0 by adding 51 µl of 0.5 N NaOH and added to a mixture of 50 µl carboxypeptidase-U (13.6 mg / ml) o After 15 min, the reaction is stopped by the introduction of 25 µl 6 M HC1, pH is adjusted to 1.2 "The reaction mixture is separated into Sephadex G-50 and eluted with 1 M acetic solution Acids Get 8 mg of human insulin (proven by amino acid analysis) „
    Example 3 about 5 mg of methyl ef1pha human insulin (ins-tre-OSI) is dissolved in 1 ml of 0.1 M KCl with 1 mM EDTA with an increase in pH from 4.5 to 8, -5 by the addition of NaOHo. This pH range is kept in pH-state using 0.05 M NaOH as a titrant at room temperature. The reaction is started by adding 1 µg of carboxypeptidase yeast dissolved in 10 µl of water. The progress of the reaction is monitored by high-performance liquid chromatography. After 1 h 10 min. The reaction mixture is gel filtered in 30% yKcycHoii acid on Sephadex. the course, determined by A ,, was 4.8 mg of human insulin,
    Amino acid analysis of the composition is given in tabLo 2 about
    table 2
    The invention allows to simplify the conventional method of obtaining human insulin and shorten the time of its implementation by reducing LTHCJIH labor-intensive Formula Operators and 3 products.
    The method of obtaining human insulin by treating the insulin derivatives with carboxypeptidase, followed by isolation of the target product, about t and t that with the aim of simplifying the method, 1c-tri-NHg, HHc- is also used as insulin derivatives Tpe-Tpe-mig or Ins-Tre-OCH where „ns-des-tre (B 30) -insulin is a subtle, and as carboxypeptidase it is a yeast carboxypeptidase-Y, and the processing is carried out in solution or dispersion at pH 7.5-10.0.
    权利要求:
    Claims (1)
    [1]
    Claim
    A process for preparing human insulin by treatment with carboxypeptidase insulin derivatives, followed by separation fission product of L and t n and w u n d I with the fact that, in order to simplify the process, as insulin derivatives are used ins treΝΗ-2-ins tre- Tre-Sh 2 or Ins-Tre-0СН 3 where ins-des-tre (H 30) is human insulin, and as carboxypeptidase yeast carboxypeptidase-Υ, and the treatment is carried out in Persia solution or bottom at pH 7.5-10 0 ”
    类似技术:
    公开号 | 公开日 | 专利标题
    SU1611217A3|1990-11-30|Method of producing human insulin
    FLEER et al.1978|The primary structure of bovine pancreatic phospholipase A2
    Brady et al.1979|Practical synthesis of cyclic peptides, with an example of dependence of cyclization yield upon linear sequence
    VÉRTESY et al.1984|Tendamistat |, a tight‐binding α‐amylase inhibitor from Streptomyces tendae 4158: Isolation, biochemical properties
    FI112116B|2003-10-31|A method for determining the 3-dimensional structure information of proteins and a nutrient medium
    DE69731105T2|2005-11-17|METHOD FOR SELECTIVE ACETYLATION
    CA1172978A|1984-08-21|.alpha.-AMYLASE INACTIVATOR, A PROCESS FOR ITSPREPARATION, AN AGENT BASED ON THIS INACTIVATOR ANDITS USE
    Li et al.1970|The amino acid sequence of hemoglobin V from the lamprey, Petromyzon marinus
    Takagaki et al.1980|Amino acid sequence of the membranous segment of rabbit liver cytochrome b5. Methodology for separation of hydrophobic peptides.
    Matthews et al.1972|Evidence for the presence of two nonidentical subunits in carbamyl phosphate synthetase of Escherichia coli
    US4644057A|1987-02-17|Process for the cleavage of peptides and proteins at the methionyl bond using cyanogen chloride
    KR19990023651A|1999-03-25|How to Obtain Insulin Precursor with Correctly Bound Cystine Bridges
    EP1448786B1|2010-04-14|Process for preparing insulin compounds
    GB2069502A|1981-08-26|Process for Preparing Insulin Esters
    EP0017938B1|1983-08-03|Process for preparing a b30-threonine insulin
    CA1174973A|1984-09-25|Process for preparing polypeptide derivatives
    De Jersey et al.1969|Oxazolinone intermediates in the hydrolysis of activated N-acylamino acid esters. The relevance of oxazolinones to the mechanism of action of serine proteinases
    US20120214965A1|2012-08-23|Glargine proinsulin and methods of producing glargine insulin analogs therefrom
    WO2012115638A1|2012-08-30|Glargine proinsulin compositions and methods of producing glargine insulin analogs therefrom
    Svoboda et al.1994|Semisynthetic insulin analogues modified in positions B24, B25 and B29
    CS271191A3|1992-03-18|Enzymatic process of pre-proinsulins to insulins conversion
    EP0085083B1|1985-01-23|Process for the enzymatic preparation of human insulin
    DK173007B1|1999-11-08|Process for the semi-synthetic preparation of human insulin and modified human insulin for use in the process
    US20160024168A1|2016-01-28|Aspart proinsulin compositions and methods of producing aspart insulin analogs
    US7396903B2|2008-07-08|Process for preparing insulin compounds
    同族专利:
    公开号 | 公开日
    HU185668B|1985-03-28|
    ES504462A0|1982-12-01|
    ES8300858A1|1982-12-01|
    EP0045187B1|1984-10-10|
    AU7450881A|1982-02-16|
    AU543687B2|1985-04-26|
    ZA815066B|1983-03-30|
    SU1554766A3|1990-03-30|
    CA1175372A|1984-10-02|
    JPS57501161A|1982-07-08|
    DE3166608D1|1984-11-15|
    AT9820T|1984-10-15|
    ES514463A0|1983-06-01|
    EP0045187A1|1982-02-03|
    WO1982000301A1|1982-02-04|
    JPS642360B2|1989-01-17|
    IE51620B1|1987-01-21|
    ES8306182A1|1983-06-01|
    IE811660L|1982-01-24|
    US4645740A|1987-02-24|
    DK319780A|1982-01-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US3276961A|1963-04-08|1966-10-04|Squibb & Sons Inc|Process for preparing human insulin|
    US4182654A|1974-09-18|1980-01-08|Pierce Chemical Company|Production of polypeptides using polynucleotides|
    BR8008024A|1979-04-06|1981-03-31|Forenede Bryggerier As|PROCESS FOR ENZYMATIC PRODUCTION OF PEPTIDEOS|
    JPS5746360B2|1979-04-13|1982-10-02|
    DK146482C|1979-04-13|1986-10-06|Shionogi & Co|METHOD FOR PREPARING A B30-THREONINE INSULIN|
    US4343898A|1980-02-11|1982-08-10|Novo Industri A/S|Process for preparing esters of human insulin|DE3101382A1|1981-01-17|1982-09-02|Hoechst Ag, 6000 Frankfurt|"METHOD FOR PRODUCING HUMANISULIN OR ITS DERIVATIVES FROM PIG INSULIN OR ITS DERIVATIVES"|
    WO1982004069A1|1981-05-20|1982-11-25|Andresen Finn Hede|A process for the preparation of insulin derivatives|
    DK353781A|1981-08-10|1983-02-11|Novo Industri As|PROCEDURE FOR THE PREPARATION OF INSULIN DERIVATIVES|
    AU551174B2|1981-09-15|1986-04-17|Nordisk Insulinlaboratorium|Enzymatic preparation of human insulin or b-30 esters thereof|
    EP0087238A1|1982-02-08|1983-08-31|Biogen N.V.|Am improved method for preparing human insulin from non-human insulin|
    DE3209184A1|1982-03-13|1983-09-15|Hoechst Ag, 6230 Frankfurt|METHOD FOR CONVERTING PRAEPROINSULIN ANALOGS TO INSULINES|
    NL8201650A|1982-04-21|1983-11-16|Akzo Nv|SEMISYNTHETIC PREPARATION OF HUMANE INSULIN.|
    DE3326472A1|1983-07-22|1985-02-14|Hoechst Ag, 6230 Frankfurt|NEW INSULIN DERIVATIVES, METHOD FOR THE PRODUCTION AND USE THEREOF AND PHARMACEUTICAL AGENTS FOR TREATING THE DIABETES MELLITUS|
    DE3327709A1|1983-07-29|1985-02-07|Hoechst Ag, 6230 Frankfurt|INSULIN DERIVATIVE CRYSTAL SUSPENSIONS, METHOD FOR THE PRODUCTION AND USE THEREOF|
    DE3333640A1|1983-09-17|1985-04-25|Hoechst Ag, 6230 Frankfurt|METHOD FOR THE PRODUCTION OF INSULIN DERIVATIVES, THE B-CHAIN C-TERMINAL EXTENDED, NEW BASICALLY MODIFIED INSULIN DERIVATIVES, THE MEANS CONTAINING THEM AND THEIR USE|
    DK113585D0|1985-03-12|1985-03-12|Novo Industri As|NEW PEPTIDES|
    DK119785D0|1985-03-15|1985-03-15|Nordisk Gentofte|INSULIN PREPARATION|
    PH25772A|1985-08-30|1991-10-18|Novo Industri As|Insulin analogues, process for their preparation|
    DK220890D0|1990-09-14|1990-09-14|Ole Buchardt|PROCEDURE FOR THE PREPARATION OF C-TERMINALLY AMIDATED PEPTIDES|
    US5580751A|1990-09-14|1996-12-03|Carlsberg A/S|Process for the preparation of C-terminally amidated peptides|
    US6869930B1|1993-09-17|2005-03-22|Novo Nordisk A/S|Acylated insulin|
    US6011007A|1993-09-17|2000-01-04|Novo Nordisk A/S|Acylated insulin|
    CA2171424C|1993-09-17|2002-06-04|Svend Havelund|Acylated insulin|
    AU1112597A|1996-07-25|1998-02-20|Firma "Nika-Universal"|Binary techniques for producing biologically active peptides|
    US6323311B1|1999-09-22|2001-11-27|University Of Utah Research Foundation|Synthesis of insulin derivatives|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DK319780A|DK319780A|1980-07-24|1980-07-24|PROCEDURE FOR ENZYMATIC REPLACEMENT OF B-30 AMINO ACID IN INSULINES|
    [返回顶部]