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    • 专利摘要:
    A process for preparing an insulin precursor is described, of the formula <CHEM> in which R is hydrogen, a chemically or enzymatically cleavable amino acid residue, or a chemically or enzymatically cleavable peptide moiety having at least two amino acid residues; Y is <CHEM> in which Z is Ala, Thr, or Ser; the moiety from A-1 to A-21 is an insulin A-chain; the moiety from B-1 to B-30 is an insulin B-chain; and X is a moiety which is joined to the insulin A-chain at the amino group of A-1 and to the insulin B-chain at the epsilon -amino group of B-29 or the carboxy group of B-30, which moiety can be enzymatically or chemically cleaved from and without disruption of both the A-chain and the B-chain, which comprises reacting an S-sulfonate of the formula <CHEM> in which R, X, Y are as defined above, with a mercaptan in an amount which provides from about 1 to about 5 -SH moieties per each -SSO3- moiety in an aqueous medium at a pH of from about 7 to about 11.5 and at an S-sulfonate concentration of up to about 10 mg. per ml. of aqueous medium.
    公开号:SU1301319A3
    申请号:SU813264049
    申请日:1981-03-26
    公开日:1987-03-30
    发明作者:Хилл Франк Брюс
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    one
    This invention relates to a method for producing bovine, porcine or human proinsulin, a biologically active compound used in biology and medicine.
    The purpose of the invention is to simplify the process.
    Getting the original products.
    Linear chain S-sulfonate precursor of insulin.
    786 mg of sodium sulfite is added to I00 ml of cooled deionized 7M urea, the solution is stirred, 594 mg of sodium tetrathionate is added, stirring is continued, however the solution remains cloudy. The pH is adjusted to 7.7 with glacial acetic acid. HPZC purified bovine proinsulin (503 mg) is added with stirring. The pH of the reaction solution is adjusted to 7.6 2n. sodium hydroxide. The obtained turbid solution was stirred at 6 ° C for 18 h.
    Approximately half of the reaction mixture is separated, adjusted to pH 9.1 with 2N. sodium hydroxide, and injected onto a Sephadex M-25 column (coarse).
    Condition chromatography: solvent - 0.05 M ammonium bicarbonate; pH 9.0; column size 2x90 cm; temperature 21 C; flow rate 18.5 ml / min.
    The first 120 ml of the effluent is drained and the next 75 mp is collected. Then the column is washed with another portion of 0.05 M ammonium bicarbonate at pH 9.0 (AOO ml). This procedure is repeated for the second half of the reaction solution. The UV spectra of the two pools show that only 401 mg was obtained. The pools were combined and lyophilized to dryness. 445.7 mg of dry desalted product are obtained. The structure of the obtained product - linear chain S-sulphonate bovine proinsulin and the absence of starting material was confirmed by cellulose acetate electrophoresis and polyamide disk gel electrophoresis.
    Linear chain S-sulfonate bovine proinsulin is purified by chromatography on DEAE-CellShose. The crude sample (443 mg) was dissolved in a 10 mp mixture of 7.5 M urea — 0.0% Tris – 0.001 M EDTA at pH 8.5 and introduced into a DEAE-cellulose column.
    013192
    Chromatography conditions: solvent — 7.5 M urea — 0, Tris — 0.001 M EDTA, pH 8.5 with a gradient of 0-0.35 M sodium chloride; column size 5 2.5x90 cm; temperature 4 ° C, flow rate about 0.9 ml / min; fraction volume 5.3 ml.
    Absorption at 276 nm for each fraction, constructed depending on JO on the fraction number, indicates a large peak with a small tail, UV spectroscopy showed that a large peak is the product obtained, fractions 199-240 with the volume of the leaked phase 15 1069-1291 ml unite. The UV spectrum for this sample is 355 mg.
    The resulting pool desalted on a column with Sephadex M-25 (coarse). 20 Chromatography conditions:
    the solvent is 0.05 M ammonium bicarbonate; pH 8.0; column size 3.7x x105 cm, temperature 4 ° C; speed
    25
    flow 16.0 ml / min.
    The first 396 ml of liquid is drained, and the next 250 ml is collected and stored. Then the column is washed with another portion of 2000 mp 0.05 M bicarbon 30 ammonium with a pH of 8.0.
    The UV spectrum of this pool showed 321 mg of this sample. The sample is lyophilized to dryness. Collect 373 mg of dry material. The identity of the product is confirmed by polyacrylamide disk gel electrophoresis and high performance low pressure HPZPZC liquid chromatography based on the elution time.
    40 In the following, their examples describe the production of bovine, porcine or human proinsulin.
    Example 1 Concentration 0.1 mg / ml.
    45 To a solution of 1.61 mg of linear chain S-sulphonate bovine proinsulin in 16.1 ml of degassed 0.05 M glycine (pH 9.5) was added 0.158 ml of aqueous 2-mercaptoethanol rast50, which, when titrated with Ellman's reagent, showed the content of mercaptan 2 , 11 mg / ml, which corresponds to 4 eq. 2-mercaptoethanol on - SSO - in the linear chain Z-sulfo55 natno bovine proinsulin. The final pH value is 9.46. The solution prepared at room temperature, sealed with paraffin, and then stirred while cooling to 6 C for 19 hours
    The reaction mixture is then acidified to pH 4.0 ± 0.1 (thermostatically) using concentrated hydrochloric acid and 0.5N. sodium hydroxide. The product is isolated. The structure was confirmed by high pressure liquid chromatography (HPZPZC).
    The conditions of HPZPZC are as follows: column 1.1x54 cm, glass column filled with LP-1 / C, with C content of 16.6%; the solvent is 30% acetonitrile and 70% ammonium formate, pH 4.25; temperature 21 C; pressure 8028 flow rate 2.40 ml / min.
    The sample is introduced into the column using 5 units of a test loop-like injector and operates at a wavelength of 280 nm.
    The first sample introduced was 5 ml of bovine insulin solution with a nominal protein concentration of 0.1 mg / ml; the second introduced sample is 5 ml of the acidified reaction mixture. The presence of monomeric bovine proinsulin in the reaction mixture was confirmed based on the elution time. The calculation of the peak areas of the two HPZPZC cycles showed that the yield of bovine proinsulin in the reaction mixture was 82.6%.
    The research results are presented in Table. one.
    Table 1
    Asp thr Ser Glu Tro Gly Ala
    Continuation of table 1
    ten
    15
    20
    thirty
    25 Residual values are the timelines for three determinations (30 hours of hydrolysis), Ala 1.0.
    Example 2 Concentration 0.5 ml / mg
    To a solution of 25.07 mg of linear chain S-sulphonate bovine. Proinsulin in 50.14 ml of degassed 0.05 M glycine, pH 10.51, was added 1.302 ml of an aqueous 2-mercaptoethanol solution, which during the titration with Ellman's reagent has a concentration of mercaptan 2.10 mg / ml, which corresponds to 2.1 eq. 2-mercaptoethanol on - SSO - in linear chain-40 S-sulphonate bovine proinsulin. The final pH is 10.47. The solution prepared at room temperature, sealed with paraffin and then stirred while cooling to 45 to 6 C for 18 hours
    The reaction mixture is acidified to pH 4.010.1 (thermostatic control) using concentrated hydrochloric acid and 0.1N. hydrochloric acid. Analysis using HPZPZC prkgazal 69% yield of bovine proinsulin in the reaction mixture.
    The resulting product, after bleaching, is isolated by gel J5 filtration chromatography. The pH of the reaction mixture was adjusted to 9.0 with concentrated ammonium hydroxide and introduced into a column filled with Sephadex Zh-25.
    50
    five.
    Conditions for chromatographic desalination: solvent — 0.05 M ammonium bicarbonate, pH 9.0; column size 2x90 cm; temperature 21 ° C; flow rate 18.5 mp / min.
    The first 120 mp of the liquid obtained are drained, the next 75 are collected (protein pool). Then the column is washed with another portion of 400 ml of 0.05 M ammonium bicarbonate with a pH of 9.0,
    The UV spectrum of the protein pool showed that 21.6 mg of protein was absorbed. This pool is lyophilized by hocyxa. 22.21 mg of dry desalted protein are obtained.
    A part of this material (14.94 mg) is dissolved in 5.5 mp 1, OM acetic acid,
    The UV spectrum of the clear solution showed that the protein concentration was 2.56 mg / ml. 5 ml of this solution (12.8 ml according to UV data) are introduced into the column; With Sephadex Zh-50 (ultrathin),
    Chromatography conditions: solvent: IM acetic acid; column dimensions 1,5x100 cm temperature 21 ° С; flow rate 0.19 ml / min. fraction volume about 1.9 ml.
    As elution was effected overnight, IM acetic acid recorded the absorbance at 280 nm. On the resulting curve - two peaks. The first, smaller peak corresponds to the aggregate-, ny forms of bovine proinsulin; the second peak is monomeric bovine insulin. Pools are collected for two peaks. The fractions obtained are combined,
    Pool I: fractions 30-46 (55.0-84.0 m peak 70.4 ml),
    Pool II: fractions 47-62 (84.0-112.0 ml; peak 99.8 mp).
    According to UV spectra, pool I contains 1.94 mg, pool 10 contains 10, and t, e, total 12.05 mg (yield 94.1% of the amount injected into the column), total 83.9 % monomeric bovine insulin.
    Both pools are lyophilized to dryness. pool I identified as bullish | Proinsulin based on time and elution in the HPZPZC cycle, which was also confirmed by trypsin and carboxypeptidase B treatment (Table 2),
    6
    Table 2
    Example 3. Effect of temperature.
    The procedure of Example 1 was used to determine the effect of temperature on the release of bovine insulin from linear chain S-sulphonate bovine proinsulin.
    Reaction conditions: concentration; protein 0.1 ml / mg; buffer - 0.05 M glycine; pH 9.5; mercaptan - 2-mercaptoethanol in an amount that provides 4 eq. - SH on - SSOj-; time 18 h
    Carrying out the reaction at 21 ° C provides a yield of 47% proinsulin
    71
    According HPZPZC, If the reagents are mixed at 21 ° C, and then the process is carried out with cooling to 6 C, the yield is 77%.
    Example 4, pH effect.
    The procedure of Example 1 is used to determine the effect of pH on the yield of bovine proinsulin from linear chain S-sulphonate bovine proinsulin in a number of reactions carried out simultaneously.
    Reaction Conditions: protein concentration 0.5 mg / ml; buffer - 0.05 M glycine; mercaptan - 2-mercaptoethanol in an amount that provides 2 eq, - SH on - ZZOZ; time 18h; temperature 6 C.
    According to HPZPZC, the proinsulin yield as follows is pH dependent:
    pH Yield,% 9.0 9.5 10.0 10.5 11.0
    Pool I - 22.1
    Pool III — 103.6 mg, which is 154 mg and corresponds to a yield of 94% of the amount introduced into the column. Of the amount recovered, 63.7% is monomeric human pro insulin. All three pools are frozen and lyophilized to dryness.
    Of the three pools, 106.55 mg of sygy material is collected. Human proinsulin is identified by amino acid analysis and polyacrylamide disk gel electrophoresis, as well as on the basis of zylyuvani on LC pH at which the release of human proinsulin in relation to bovine proinsulin is expected. Further identification (| ikatsiya carried out when processing trypsin and carboxypeptidase B to obtain human insulin (Table, 3),
    Table 3
    43.1 44.3 66.7 76.0 61.0 mg; pool II -28.3 mg;



    Asp
    4.07
    4.0
    Thr
    2., 82
    3.0
    8 Continuation of table 3
    20
    25
    thirty
    Residual values are average of three determinations (30 hours of hydrolysis), Ala 1.0,
    Example 5, Effect of protein concentration on product yield.
    The procedure of Example 1 is used to determine the effect of protein concentration on the yield of bovine proinsulin from linear chain S-sulphonate bovine proinsulin in the p-de reactions carried out simultaneously.
    Reaction conditions: buffer - 0.05 M glycine; pH 9.5; mercaptan - 2-mercaptoethanol in quantity, provide
    vayuschim 4 equiv, - SH for - time 18 h; temperature 6 ° С
    According to HRZPZC, the change in protein concentration in the following way affects the yield of proinsulin:
    Concentration of pro- Yield,% of theine, mg / ml
    0.1
    78
    0.2
    63

    46
    37.6 25.4 42
    When conducting another cycle of experiments for 2 eq. - SH on - SSO.J - and at pH 10.5, the following results are obtained:
    Protein concentration, mg / ml
    0.5
    0.96
    1.83
    4.2
    7.4
    Output, %
    77.2 58.3 19.5
    G5
    According to HPZPZC, the type of mercapt affects the output of proinsulin as follows:
    20
    Mercaptan
    Dithiothreitol
    Dithioerythritol
    Exit, 39.3 34.9
    20.1 Methylthioglycol T56.1
    19.6 H-Mercapto-1,2-propandiol 65.5
    Moreover, in the last two cases, the ratio SH: - SSOj - 1.2.
    Example 6. The effect of the ratio - SH: - SSOg - on the output of proinsulin.
    The procedure of Example 1 is used to determine the effect of the ratio — SH.c — SSOj — on the output of bovine proinsulin from linear chain S-sulphonate bovine proinsulin in a series of reactions carried out simultaneously.
    Reaction Conditions: Protein concentration 0.5 mg / ml; buffer - 0.05 M glycine; pH 9.5; time 18h; temperature .
    According to HPZPZC, a change in the ratio — SH: - SSO — affects the output of proinsulin as follows:
    Ratio Yield%
    Example 7. Effect of mercaptan type on proinsulin yield.
    130131910
    The procedure of Example 1 is used to determine the effect of the structure of a mercaptan on the yield of bovine proinsulin from a linear chain S-sulfate phonate bovine proinsulin in a series of reactions carried out simultaneously.
    Reaction Conditions: protein concentration 0.1 mg / ml; buffer - 0.05 M glycine; pH 9.5; mercaptan, 4 eq. - SH on - SSOj-; time 18 h; temperature 6 C,
    According to HPZPZC, the type of mercaptan affects the yield of proinsulin as follows:
    Mercaptan
    Dithiothreitol
    Dithioerythritol
    Yield,% 39.3 34.9
    3-Mercaptopropionov
    acid65,3
    2-Mercaptoethanol64.1
    Example 8. The effect of protein type on the yield of proinsulin.
    The procedure of example 1 is used
    to determine the effect of protein type on the output of proinsulin from linear chain S-sulphonate proinsulin in a series of reactions carried out simultaneously.
    Reaction Conditions: Protein concentration .0.1 mg / ml; buffer - 0.05 M shlycine; pH 9.5; mercaptan - 2-mercaptoethanol in an amount that provides 4 eq. - SH on - SSOj-; time 18 h; temperature 6 C.
    According to HPZPZC, the proinsulin yield as follows depends on the type of protein:
    Linear chain S-sulfonate proinsulin
    Bull Pork
    %
    60.6 65.8
    Example 9. Obtaining human insulin.
    To a solution of 169.3 mg of biosynthetically prepared linear chain S-sulphonate human proinsulin in 338.6 ml of degassed 0.05 M glycine (, 54) was added 7.71 MP of an aqueous solution of 2-mercapto-ethanol, which during the titration Ellman's reagent showed a mercaptan concentration of 2.08 mg / ml, which corresponds to 2 eq. 2-mercaptoethanol on JQ - SSO - in linear chain S-sulphonate human proinsulin. Using 5N sodium hydroxide, the pH was adjusted to 10.52. The solution is sealed with paraffin and stirred at 6 ° C J5 for 18 hours.
    Then the reaction mixture is acidified to pH 2.9 + 0.1 (thermoregulation) with concentrated hydrochloric acid. The resulting clear solution was introduced 20 into a column filled with Sephadex G-25 (coarse) for desalting.
    Conditions of chromatography: solvent - 2% acetic acid; column dimensions 5x100 mm; temperature is 25 ° C; flow rate 28.8 ml / min; fraction volume 20.2 ml.
    The first 789 ml of the obtained liquid is drained, and the next 464 mp is collected and stored. The determination of optical density at 280 nm showed that it is a protein pool. The column is washed with an additional 2500 ml of 2% acetic acid. Calculations based on the human form of the second peak — monomerically co-insulin (part shoulders). Three n fractions are collected. Fractions are combined.
    Pool 1: fractions 46-67 pool II: fractions 68.81 (3 pool III: fractions 82,490.3 ml) ..
    For these pools, the following proteins are calculated:
    pool I - 22.1 mg; Pu Pool III - 103.6 mg.
    Received 154 mg corresponds to a yield of 94% va, entered in a column amount of 67.3% numbered human All 3 pools are frozen to dryness.
    From three pools collect dry material.
    Amino acid anal amide disk gel confirmed that he received cue proinsulin. Time does not correspond to human elution with respect to bovine when HPZC is used. Further identification d t when treating trip oxypeptidase B (up to
    nyu UV spectrum data for protein and gland insulin).
    Go to the pool showed that the yield of protein is 164 mg, which corresponds to 101.9% of the amount introduced into the column (theoretical yield of the reformation reaction). This pool is frozen and lyophilized to dryness.
    The desired product is isolated using gel- (| chromatography). The dry product (not weighed) is dissolved in 20 ml of W of acetic acid. The resulting clear solution is introduced into a column filled with Sephadex Ж-50 (ultrathin).
    Condition chromatographic: solvent - 1M acetic acid; column dimensions 2,5x125 cm; temperature 25 ° С; flow rate 0.82 ml / min; fraction volume about 4.92 ml.
    The column was eluted overnight with acetic acid; Absorbance at 270 nm was recorded. On the obtained absorption curve at 280 nm, 2 peaks were obtained from the fraction number. First peak (smaller) corresponds to aggregate40
    Example 10. Che pro insulin cysteine,
    To a solution of 130 mg h of proinsulin-8-sulfonate dehydrated cycin with a pH of 10.4, added an initial aqueous plant that has 3.63 mg / ml (titration, Ellman) contained, which corresponds to 50 wt.% Mercapt pH 10.5 reaches from a lazy small amount of sodium rooxide. The solution is stirred at 6 ° C.
    50
    55
    Thereafter, the reaction is acidified to a pH of 3.8 with aqueous acid. According to Dan1 high chromatography (HPLC), the yield was 56.3 wt.%. Wine is added to the acidification mixture to obtain a urea endpoint of 4
    In the form of human proinsulin, the second peak, monomeric human proinsulin (anterior shoulder), three pools of fractions are collected. Fractions are combined.
    Pool 1: fractions 46-67 (218-325.5 ml); pool II: fractions 68.81 (325, 6-395.5 ml); pool III: fractions 82-100 (395.5- 490.3 ml) ..
    The following amounts of protein were calculated for these pools according to UV spectra:
    pool I - 22.1 mg; pool II - 28.3 mg; pool III - 103.6 mg.
    154 mg of product was obtained, which corresponds to a yield of 94% of the amount introduced into the column. Of the amount recovered, 67.3% is monomeric human proinsulin. All 3 pools are frozen and lyophilized to dryness.
    Of the three pools, 106.55 mg of dry material is collected.
    Amino acid analysis and polyacryl amide disk gel electrophoresis confirmed that human proinsulin was obtained. The time of its eluting under HPZC corresponds to the elution time of human proinsulin in relation to bovine proinsulin. Further identification is carried out when treating with trypsin and carboxypeptidase B (until receipt of
    Example 10. Human proinsulin-cysteine,
    To a solution of 130 mg of human proinsulin-8-sulfonate in 404 mp of cold degassed 0.02 M glycine with a pH of 10.4, 13.9 ml of the original aqueous cysteine solution, which has a mercaptan content of 3.63 mg / ml (titration with a reagent Ellman), which corresponds to the content of 50 wt.% Mercaptan in protein. A pH of 10.5 is reached by adding a small amount of 5N sodium hydroxide. The solution is closed and stirred at 6 ° C for 18 hours.
    0
    five
    The reaction mixture is then acidified to pH 3.8 with glacial acetic acid. According to 1m high pressure liquid chromatography (HPLC), the yield of proinsulin is 56.3 wt.%. Solid urea is added to the acidified reaction mixture and a final urea concentration of 4M is obtained. Proinsulin
    13.1
    is isolated by ion exchange chromatography on SP-Sephadex, and then by chromatography on DEAE. The resulting purified compound is then purified by gel filtration chromatography (} on Sephadex G 50SF. The compound obtained is human proinsulin, which is confirmed by HPLC with simultaneous elution of human proinsulin as a comparison. Yield of solid human proinsulin 43 wt.%. 12 mg human Proinsulin is selected for analytical purposes during the described filtration (if this amount is included in the resultant yield, then the total yield of proinsulin will be 52 wt.%).
    The research results are summarized in table. four.
    Table 4
    Asp Thr Ser Glu Pro Gly Ala Cys Val Jle Leu Tyr Phe His Lys Yrg
    1914
    Residual values are averages for three determinations (30 hours of hydrolysis). Ala 1.0.
    The proposed method allows directly, in one stage, to convert the S-sulfonate to the target disulfide precursor of insulin due to the direct exchange under conditions that do not require the presence of oxygen.
    权利要求:
    Claims (2)
    [1]
    Invention Formula
    1, Method for preparing bovine, porcine or human proinsulin of general formula 1
    (A-1 G19 - NH
    (A-6) Cys-S-S (A-gO) (/ 1-21) (A-7) CijsCyS-Ci) S-ASn-OH
    S (A-IO S
    SS
    (B-1 R-WH-Phe-Cys
    -Cgs
    where R is hydrogen;
    Y is Lys-Ala- or -Lys-Thr-; X - Arg-Arg-Glu-Val-Glu-Gly-Pro-Gln-Val-Gly-Ala-Leu-Glu- -Ceu-Ala-Gly-Gly-Pro-Gly-Ala-Gly-Gly-Leu- Glu-Gly-Pro-Pro-Gln-Lys-Arg; Arg-Arg-Glu-Ala-Glu-Asn-Pro-Gln-Ala-Gly-Ala-Val-Glu-Leu-Gly-Gly-Gly-Leu-Gly-Gly-Leu-Gln-Ala-Leu -Ala- -Leu-Glu-Gly-Pro-Pro-GIn- -Lys-Argt
    -Arg-Arg-Glu-Ala-Glu-Asp- -Leu-Glu-Val-20Gly-Gln-Val- -Glu-Leu-Gly-Gly-Gly-Pro- -Gly-Ala-Gly-SerrLeu-Glu- -Pro-Leu-Ala-Leu-Ala-Leu- -Glu-Gly-Ser-Leu-Glu-Lys- -Arg-,
    by converting S-sulfonate groups
    8-sulfonate) 1x absch polypeptides
    Formula II
    U-1 GUJ - NH-X
    (Ab) Cys-S-Sof S-SO
    I1 (A-20 (A-21)
    (jySCijS-Ci) S-Asn-OH
    S-SO k-SO
    tA-7)
     3
    s-sof
    R-HH-Phc-Cys (B-11
    f-sdf
    (B-7)
    -Suz- (BH9)
    15
    where R, X and Y have the indicated meanings,
    into disulfide bridging groups, characterized in that, in order to simplify the process, S-sulfonate polypeptides of general formula II are reacted with mercaptan, in an amount that provides 1.0-4.0 equivalents of SH-fragment for each SSO fragment in aqueous medium at pH 9-11, the concentration of S-sulfonate polypeptide II
    1319
    sixteen
    fO
    0.1-7.4 mg per 1 ml of aqueous medium at.
    [2]
    2. The method according to p. 1, characterized in that the pH of the reaction medium is maintained by adding glycine at a concentration of 0.05M.
    Priority featured
    27.03.80 - bovine, porcine proinsulin;
    28,11,80 - human pro insulin.
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    DE2253327A1|1972-10-31|1974-05-09|Hoechst Ag|PROCESS FOR THE MANUFACTURING OF INSULIN, INSULIN ANALOGS AND DERIVATIVES|JPS58183659A|1982-03-31|1983-10-26|Jieneteitsukusu Inst|Modified proinsulin precursor, dna arrangement for coding same and production thereof|
    DK58285D0|1984-05-30|1985-02-08|Novo Industri As|PEPTIDES AND MANUFACTURING AND USING THEREOF|
    DE3501641A1|1985-01-19|1986-07-24|Hoechst Ag, 6230 Frankfurt|METHOD FOR OBTAINING INSULIN PRECURSORS FROM REACTION MIXTURES WHICH ARE INCLUDED IN THE FOLDING OF INSULIN PRECURSORS FROM THE CORRESPONDING S-SULPHONATES|
    DK336188D0|1988-06-20|1988-06-20|Nordisk Gentofte|propeptides|
    DE3901719A1|1989-01-21|1990-07-26|Hoechst Ag|METHOD FOR PRODUCING AN INSULIN PREVENTE|
    DE3901718A1|1989-01-21|1990-07-26|Hoechst Ag|METHOD FOR RENATURING INCORRECT RECOMBINANT OF INSULIN PREFERRED|
    DK0600372T3|1992-12-02|1997-08-11|Hoechst Ag|Process for the preparation of proinsulin with properly connected cystine bridges.|
    DE4405179A1|1994-02-18|1995-08-24|Hoechst Ag|Method of obtaining insulin with correctly connected cystine bridges|
    WO2017040363A1|2015-09-02|2017-03-09|Merck Sharp & Dohme Corp.|A process for obtaining insulin with correctly formed disulfide bonds|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US13438980A| true| 1980-03-27|1980-03-27|
    US21069680A| true| 1980-11-28|1980-11-28|
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